Osteoinductive demineralized cancellous bone

ABSTRACT

An osteoinductive demineralized bone matrix, corresponding osteoimplants, and methods for making the osteoinductive demineralized bone matrix are disclosed. The osteoinductive demineralized bone matrix may be prepared by providing demineralized bone and altering the collagenous structure of the bone. The osteoinductive demineralized bone matrix may also be prepared by providing demineralized bone and compacting the bone, for example via mechanical compaction, grinding into a particulate, or treatment with a chemical. Additives such as growth factors or bioactive agents may be added to the osteoinductive demineralized bone matrix. The osteoinductive demineralized bone matrix may form an osteogenic osteoimplant. The osteoimplant, when implanted in a mammalian body, may induce at the locus of the implant the full developmental cascade of endochondral bone formation including vascularization, mineralization, and bone marrow differentiation. The osteoinductive demineralized bone matrix may also be used as a delivery device to administer bioactive agents.

BACKGROUND

This application claims benefit of U.S. Ser. No. 60/944,417, filed onJun. 15, 2007 and U.S. Ser. No. 60/986,843 filed on Nov. 9, 2007, eachof which are hereby incorporated by reference in their entireties.

Introduction

Mammalian bone tissue is known to contain one or more proteinaceousmaterials, presumably active during growth and natural bone healing,that can induce a developmental cascade of cellular events resulting inendochondral bone formation. The active factors variously have beenreferred to in the literature as bone morphogenetic or morphogenicproteins (BMPs), bone inductive proteins, bone growth or growth factors,osteogenic proteins, or osteoinductive proteins. These active factorsare collectively referred to herein as osteoinductive factors.

It is well known that bone contains these osteoinductive factors. Theseosteoinductive factors are present within the compound structure ofcortical bone and are present at very low concentrations, e.g., 0.003%.Osteoinductive factors direct the differentiation of pluripotentialmesenchymal cells into osteoprogenitor cells that form osteoblasts.Based upon the work of Marshall Urist as shown in U.S. Pat. No.4,294,753, issued Oct. 13, 1981, proper demineralization of corticalbone exposes the osteoinductive factors, rendering it osteoinductive, asdiscussed more fully below.

Overview of Bone Grafts

The rapid and effective repair of bone defects caused by injury,disease, wounds, or surgery has long been a goal of orthopaedic surgery.Toward this end, a number of compositions and materials have been usedor proposed for use in the repair of bone defects. The biological,physical, and mechanical properties of the compositions and materialsare among the major factors influencing their suitability andperformance in various orthopaedic applications.

Autologous cancellous bone (“ACB”) long has been considered the goldstandard for bone grafts. ACB is osteoinductive and nonimmunogenic, and,by definition, it has all of the appropriate structural and functionalcharacteristics appropriate for the particular recipient. Unfortunately,ACB is only available in a limited number of circumstances. Someindividuals lack ACB of appropriate dimensions and quality fortransplantation, and donor site pain and morbidity can pose seriousproblems for patients and their physicians.

Much effort has been invested in the identification and development ofalternative bone graft materials. Urist has published seminal articleson the theory of bone induction and a method for decalcifying bone,i.e., making demineralized bone matrix (DBM). Urist M. R., BoneFormation by Autoinduction, 150 Science 698, 893-899 (1965); Urist M. R.et al., The Bone Induction Principle, 53 Clin. Orthop. Rel. Res. 243-283(1967). As mentioned above, it is known that DBM that is derived fromcortical bone is an osteoinductive material, in that it induces bonegrowth when implanted in an ectopic site of a rodent, owing to theosteoinductive factors contained within the DBM. It is also known thatthere are numerous osteoinductive factors, e.g., BMP 1-15, which arepart of the transforming growth factor-beta (TGF-beta) superfamily BMP-2has become the most important and widely studied of the BMP family ofproteins. There are also other proteins present in DBM that are notosteoinductive alone but still contribute to bone growth, includingfibroblast growth factor-2 (FGF-2), insulin-like growth factor-I and -II(IGF-I and IGF-II), platelet derived growth factor (PDGF), andtransforming growth factor-beta 1 (TGF-beta. 1).

DBM implants have been reported to be particularly useful (see, forexample, U.S. Pat. Nos. 4,394,370, 4,440,750, 4,485,097, 4,678,470, and4,743,259; Mulliken et al., Calcif Tissue Int. 33:71, 1981; Neigel etal., Opthal. Plast. Reconstr. Surg. 12:108, 1996; Whiteman et al., J.Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin. Orthop. 293:360, 1993,each of which is incorporated herein by reference). DBM typically isderived from cadavers. The bone is removed aseptically and treated tokill any infectious agents. The bone is particulated by milling orgrinding, and then the mineral component is extracted by variousmethods, such as by soaking the bone in an acidic solution. Theremaining matrix is malleable and can be further processed and/or formedand shaped for implantation into a particular site in the recipient.Demineralized bone prepared in this manner contains a variety ofcomponents including proteins, glycoproteins, growth factors, andproteoglycans. Following implantation, the presence of DBM inducescellular recruitment to the site of injury. The recruited cells mayeventually differentiate into bone forming cells. Such recruitment ofcells leads to an increase in the rate of wound healing and, therefore,to faster recovery for the patient.

It is generally accepted that cancellous demineralized bone matrix doesnot have osteoinductive capacity. One study found that allogeneiccancellous bone blocks, demineralized or not, have no osteoinductivecapacity and no osteoconductive function that promotes healing ofmid-diaphyseal bone defects in dogs. Schwarz et al., Arch Orthop TraumaSurg., 1991; 111(1):47-50; see also Nade et al., J Bone Joint Surg Br1997 May 59(2):189-96.

Some studies indicate that the osteoinductive capabilities ofdemineralized bone from higher order species in higher order species isrelatively low. One study compared the osteoinductivity of rat andcanine bone matrix. The study looked at rat cortical bone matrix, caninecortical bone matrix, and canine cancellous bone matrix. Cortical ratbone matrix consistently induced new bone and high phosphatase levelswhen implanted ectopically in rat. Canine matrix induced small amountsof bone and lower phosphatase levels when implanted in dog and in rat,with cortical matrix being somewhat more inductive than cancellousmatrix. Demineralized cancellous bone matrix from dog was the onlymaterial tested not showing any osteoinductivity. Schwarz et al., Acta.Orthop. Scan. 60(6):693-695, 1989.

Another study, looking at cortical bone matrix from monkeys, determinedthat monkey bone matrix induces ectopic bone formation in the athymicrat but not in adult monkeys. It was concluded that adult monkey bonematrix contains bone inductive properties but that these properties arenot sufficient to induce bone formation in adult monkey muscle sites.Aspenberg et al., J. of Orthop. Res. 9:20-25, 1991.

Yet another study evaluated bone and cementum regeneration followingguided tissue regeneration (GTR) in periodontal fenestration defects.Specifically, the adjunctive effect of allogenic, freeze-dried DBMimplant was evaluated and found to exhibit no discernible adjunctiveeffect to GTR in the defect model. The critical findings were 1) the DBMparticles remained embedded in dense connective tissue without evidenceof bone metabolic activity; and 2) limited and similar amounts of boneand cementum regeneration were observed for GTR plus DBM and GTRdefects. Caplanis et al., J Periodontal 851-856, August, 1998.

Current DBM formulations have various drawbacks. First, while thecollagen-based matrix of DBM is relatively stable, the osteoinductivefactors within the DBM matrix are rapidly degraded. The osteogenicactivity of the DBM may be significantly degraded within 24 hours afterimplantation, and in some instances, the osteogenic activity may beinactivated within 6 hours. Therefore, the osteoinductive factorsassociated with the DBM are only available to recruit cells to the siteof injury for a short time after transplantation. For much of thehealing process, which may take weeks to months, the implanted materialmay provide little or no assistance in recruiting cells. In addition tothe osteoinductive factors present within the DBM, the overall structureof the DBM implant is also believed to contribute to the bone healingcapabilities of the implant.

U.S. Pat. No. 4,563,350, herein incorporated by reference in itsentirety, discloses the use of trypsinized bovine bone matrix as axenogenic matrix to effect osteogenic activity when implanted withextracted, partially purified bone-inducing protein preparations. Boneformation is said to require the presence of at least 5%, and preferablyat least 10%, non-fibrillar collagen. The named inventors claim thatremoval of telopeptides that are responsible in part for theimmunogenicity of collagen preparations is more suitable for xenogenicimplants.

European Patent Application Serial No. 309,241, published Mar. 29, 1989,herein incorporated by reference in its entirety, discloses a device forinducing endochondral bone formation comprising an osteogenic proteinpreparation, and a matrix carrier comprising 60-98% of either mineralcomponent or bone collagen powder and 2-40% atelopeptide hypoimmunogeniccollagen.

U.S. Pat. No. 3,394,370, herein incorporated by reference in itsentirety, describes a matrix of reconstituted collagen purportedlyuseful in xenogenic implants. The collagen fibers are treatedenzymatically to remove potentially immunogenic telopeptides (also theprimary source of interfibril crosslinks), and are dissolved to removeassociated noncollagenenous components. The matrix is formulated bydispersing the reconstituted collagen in acetic acid to form adisordered matrix of elementary collagen molecules that is then mixedwith an osteogenic substance and lyophilized to form a “semi-rigid foamor sponge” that is preferably crosslinked.

U.S. Pat. No. 4,172,128, herein incorporated by reference in itsentirety, describes a method for degrading and regenerating bone-likematerial of reduced immunogenicity, said to be useful cross-species.Demineralized bone particles are treated with a swelling agent todissolve any associated mucopolysaccharides (glycosaminoglycans), andthe collagen fibers subsequently dissolved to form a homogenouscolloidal solution. A gel of reconstituted fibers then can be formedusing physiologically inert mucopolysaccharides and an electrolyte toaid in fibril formation.

Various papers have looked at cartilage tissue differentiation of bonematrix gelatin (BMG) from cortical bone. Terashima and Urist found thatcortical bone BMG is chemically more reactive than whole bone matrix.Terashima et al., Chondrogenesis in Outgrowths of Muscle Tissue ontoModified Bone Matrix in Tissue Culture, 127 Clin. Orthop. and Rel. Res.248-256 (1977). A later study found that rat BMG may inducechondrogenesis in cell culture. Urist et al., Cartilage TissueDifferentiation from Mesenchymal Cells Derived from Mature Muscle inTissue Culture, 14(8) In Vitro 697-706 (1978). Nogami and Urist alsoassessed the effect of various treatments of cortical bone, includingcollagenase treatment of cortical bone BMG, on cartilage tissuedifferentiation. Nogami and Urist, Substrata Prepared from Bone Matrixfor Chondrogenesis in Tissue Culture, 62 J. of Cell Bio. 510-519 (1974).

A variety of approaches have been explored in an attempt to recruitprogenitor cells or chondrocytes into an osteochondral or chondraldefect. For example, penetration of subchondral bone has been performedin order to access mesenchymal stem cells (MSCs) in the bone marrow,which have the potential to differentiate into cartilage and bone.Steadman, et al., Microfracture: Surgical Technique and Rehabilitationto Treat Chondral Defects 391 S Clin. Orthop. 362-369 (2001). Inaddition, some factors in the body are believed to aid in the repair ofcartilage. For example, transforming growth factors beta (TGF-β) havethe capacity to recruit progenitor cells into a chondral defect from thesynovium or elsewhere when loaded in the defect. Hunziker, et al.,Repair of Partial Thickness Defects in Articular Cartilage: CellRecruitment From the Synovial Membrane, 78-A J. Bone Joint Surg.,721-733 (1996). However, the application of growth factors to bone andcartilage implants has not resulted in the expected increase inosteoinductive or chondrogenic activity.

Each of U.S. Pat. Nos. 5,270,300 and 5,041,138, each herein incorporatedby reference in its entirety, describes a method for treating defects orlesions in cartilage that provides a matrix, possibly composed ofcollagen, with pores large enough to allow cell population and containgrowth factors (TGF-β or other factors (such as angiogenesis factors))appropriate for the type of tissue desired to be regenerated.

BRIEF SUMMARY

Osteoinductive compositions, and implants and methods for theirproduction, are provided. At least partially demineralized bone matrixis treated to enhance the osteoinductive activity of the bone matrix.More specifically, the at least partially demineralized bone matrix maybe treated with enzymes, chemicals, ionizing radiation, electromagneticradiation, compaction, or other treatments to impart or enhanceosteoinductive activity of the bone matrix.

This application refers to various patents, patent applications, journalarticles, and other publications, all of which are incorporated hereinby reference in their entireties. The following documents areincorporated herein by reference in their entireties: PCT PublicationsPCT/US04/43999 and PCT/US05/003092; US Patent Application Pub. No.2003/0143258 A1; PCT Publication PCT/US02/32941; Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology (John Wiley &Sons 2002); Sambrook, Russell, and Sambrook, Molecular Cloning: ALaboratory Manual, (Cold Spring Harbor Laboratory Press 2001); Rodd,Chemistry of Carbon Compounds, vols. 1-5 and supps. (Elsevier SciencePublishers 1989); Organic Reactions, vols. 1-40 (John Wiley & Sons1991); Advanced Organic Chemistry, (John Wiley & Sons 2001). In theevent of a conflict between the specification and any of theincorporated references, the specification shall control.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description is to be regarded as illustrativein nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of Example 1.

FIG. 2 a illustrates a generally round bone particle wherein the boneparticle has been surface demineralized in accordance with oneembodiment.

FIG. 2 b illustrates an elongate bone particle wherein the bone particlehas been surface demineralized in accordance with one embodiment.

FIG. 3 illustrates formulations of compressed DBM.

FIG. 4 illustrates a formulation of FIG. 3 after 7 hours of submersionin water.

FIG. 5 illustrates a formulation of FIG. 3 after 72 hours of submersionin water.

FIG. 6 illustrates a formulation of FIG. 3 after 7 hours of submersionin water.

DEFINITIONS

Bioactive Agent or Bioactive Compound, as used herein, refers to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. In some embodiments, the bioactive agent is a growthfactor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.A more complete listing of bioactive agents and specific drugs suitablefor use in the present invention may be found in Axel Kleemann andJurgen Engel, Pharmaceutical Substances Syntheses, Patents, Applications(Thieme Medical Publishing 1999); the Merck Index: An Encyclopedia ofChemicals, Drugs, and Biologicals (Susan Budavari et al., CRC Press1996); and the United States Pharmacopeia-25/National Formulary-20(United States Pharmcopeial Convention, Inc. 2001), each of which isincorporated herein by reference in their entireties.

Biocompatible, as used herein, refers to materials that, uponadministration in vivo, do not induce undesirable long-term effects.

Bone, as used herein, refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

Demineralized, as used herein, refers to material generated by removingmineral material from tissue, e.g., bone tissue. In certain embodiments,demineralized compositions as described herein may include preparationscontaining less than 5% calcium, or less than 1% calcium by weight.Partially demineralized bone is intended to refer to preparations withgreater than 5% calcium by weight but containing less than 100% of theoriginal starting amount of calcium. In some embodiments, demineralizedbone has less than 95% of its original mineral content. Percentage ofdemineralization may refer to percentage demineralized by weight, or topercentage demineralized by depth, as described with reference to FIGS.4 a and 4 b. Surface demineralized bone is a subset of partiallydemineralized bone and refers to compositions having a demineralizedsurface and a non-demineralized core. Superficially demineralized refersto bone-derived elements possessing at least about 90 percent of theiroriginal inorganic mineral content. Partially demineralized refers tobone-derived elements possessing from about 8 to about 90 percent oftheir original inorganic mineral content. Fully demineralized refers tobone containing less than 8% of its original mineral context.Demineralized bone encompasses such expressions as “substantiallydemineralized,” “partially demineralized,” “surface demineralized,” and“fully demineralized.”

Osteoconductive, as used herein, refers to the ability of anon-osteoinductive substance to serve as a suitable template orsubstance along which bone may grow.

Osteogenic, as used herein, refers to the ability of an agent, material,or implant to enhance or accelerate the growth of new bone tissue by oneor more mechanisms such as osteogenesis, osteoconduction, and/orosteoinduction.

Osteoimplant, as used herein, refers to any bone-derived implantprepared in accordance with the embodiments of this invention andtherefore is intended to include expressions such as bone membrane, bonegraft, etc.

Osteoinductive, as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Forexample, most osteoinductive materials induce bone formation in athymicrats when assayed according to the method of Edwards et al.,Osteoinduction of Human Demineralized Bone: Characterization in a RatModel, 357 Clinical Orthopaedics & Rel. Res. 219-228 (1998),incorporated herein by reference in its entirety. In other instances,osteoinduction is considered to occur through cellular recruitment andinduction of the recruited cells to an osteogenic phenotype.Osteoinductivity score refers to a score ranging from 0 to 4 asdetermined according to the method of Edwards et al., Osteoinduction ofHuman Demineralized Bone: Characterization in a Rat Model, 357 ClinicalOrthopaedics & Rel. Res. 219-228 (1998) or an equivalent calibratedtest. In the method of Edwards et al., a score of “0” represents no newbone formation; “1” represents 1%-25% of implant involved in new boneformation; “2” represents 26-50% of implant involved in new boneformation; “3” represents 51%-75% of implant involved in new boneformation; and “4” represents >75% of implant involved in new boneformation. In most instances, the score is assessed 28 days afterimplantation. However, the osteoinductivity score may be obtained atearlier time points such as 7, 14, or 21 days following implantation. Inthese instances it may be desirable to include a normal DBM control suchas DBM powder without a carrier, and if possible, a positive controlsuch as BMP. Occasionally osteoinductivity also may be scored at latertimepoints such as 40, 60, or even 100 days following implantation.Percentage of osteoinductivity refers to an osteoinductivity score at agiven time point, expressed as a percentage of activity, of a specifiedreference score.

Proteases, as used herein, refers to protein-cleaving enzymes thatcleave peptide bonds that link amino acids in protein molecules togenerate peptides and protein fragments. A large collection of proteasesand protease families has been identified. Some exemplary proteasesinclude serine proteases, aspartyl proteases, acid proteases, alkalineproteases, metalloproteases, carboxypeptidase, aminopeptidase, cysteineprotease, collagenase, etc. An exemplary family of proteases is theproprotein convertase family, which includes furin. Dubois et al.,158(1) Am. J. of Pathology 305-316 (2001). Members of the proproteinconvertase family of proteases are known to proteolytically processproTGFs and proBMPs to their active mature forms. Dubois et al., 158(1)Am. J. of Pathology 305-316 (2001); Cui et al., 17(16) Embo J. 4735-4743(1998); Cui et al., 15 Genes & Development 2797-2802 (2001), eachincorporated by reference herein in their entireties.

Protease inhibitors, as used herein, refers to chemical compoundscapable of inhibiting the enzymatic activity of protein cleaving enzymes(i.e., proteases). The proteases inhibited by these compounds includeserine proteases, acid proteases, metalloproteases, carboxypeptidase,aminopeptidase, cysteine protease, etc. The protease inhibitor may actspecifically to inhibit only a specific protease or class of proteases,or it may act more generally by inhibiting most if not all proteases.Some protease inhibitors are protein- or peptide-based and arecommercially available from chemical companies such as Aldrich-Sigma.Protein or peptide-based inhibitors adhere to the DBM (or calciumphosphate or ceramic carrier) may provide particular benefits as theyremain associated with the matrix providing a stabilizing effect for alonger period of time than freely diffusible inhibitors. Examples ofprotease inhibitors include aprotinin, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), amastatin-HCl, alpha1-antichymotrypsin, antithrombinIII, alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF),arphamenine A, arphamenine B, E-64, bestatin, CA-074, CA-074-Me, calpaininhibitor I, calpain inhibitor II, cathepsin inhibitor, chymostatin,diisopropylfluorophosphate (DFP), dipeptidylpeptidase IV inhibitor,diprotin A, E-64c, E-64d, E-64, ebelactone A, ebelactone B, EGTA,elastatinal, foroxymithine, hirudin, leuhistin, leupeptin,alpha2macroglobulin, phenylmethylsulfonyl fluo4de (PMSF), pepstatin A,phebestin, 1,10 phenanthroline, phosphoramidon, chymostatin, benzamidineHCl, antipain, epsilon aminocaproic acid, N-ethylmaleimide, trypsininhibitor, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor, andsodium EDTA.

Stabilizing agent, as used herein, refers to any chemical entity that,when included in an inventive composition comprising DBM and/or a growthfactor, enhances the osteoinductivity of the composition as measuredagainst a specified reference sample. In most cases, the referencesample will not contain the stabilizing agent, but in all other respectswill be the same as the composition with stabilizing agent. Thestabilizing agent also generally has little or no osteoinductivity ofits own and works either by increasing the half-life of one or more ofthe active entities within the inventive composition as compared with anotherwise identical composition lacking the stabilizing agent, or byprolonging or delaying the release of an active factor. In certainembodiments, the stabilizing agent may act by providing a barrierbetween proteases and sugar-degrading enzymes thereby protecting theosteoinductive factors found in or on the matrix from degradation and/orrelease. In other embodiments, the stabilizing agent may be a chemicalcompound that inhibits the activity of proteases or sugar-degradingenzymes. According to certain embodiments, the stabilizing agent retardsthe access of enzymes known to release and solubilize the activefactors. Half-life may be determined by immunological or enzymatic assayof a specific factor, either as attached to the matrix or extractedtherefrom. Alternatively, measurement of an increase in osteoinductivityhalf-life, or measurement of the enhanced appearance of products of theosteoinductive process (e.g., bone, cartilage or osteogenic cells,products or indicators thereof), is a useful indicator of stabilizingeffects for an enhanced osteoinductive matrix composition. Themeasurement of prolonged or delayed appearance of a strongosteoinductive response will generally be indicative of an increase instability of a factor coupled with a delayed unmasking of the factoractivity.

DETAILED DESCRIPTION I. Introduction

Osteoinductive compositions and implants and methods for theirproduction are provided. According to certain embodiments, at leastpartially demineralized bone is treated with one or more enzymes,chemicals, ionizing radiation, electromagnetic radiation, or compactionto impart or enhance osteoinductive activity of the bone. The bone maybe cancellous, corticocancellous, cortical, or a composite bone. Thebone may be fully demineralized, partially demineralized, or surfacedemineralized. In some embodiments, the bone is particulated, such asthrough grinding, and the particles are compacted. Those of ordinaryskill will appreciate that a variety of embodiments or versions of theinvention are not specifically discussed below but are nonethelesswithin the scope of the present invention, as defined by the appendedclaims.

Bone is made up principally of cells, and also of collagen, minerals,and other noncollagenous proteins. Cortical bone accounts forapproximately eighty percent of skeletal bone mass. Cortical bone isstructural and bears the majority of the body's weight. Cancellous boneis relatively porous and spongy and accounts for approximately twentypercent of skeletal bone mass. Cancellous bone contains bone marrow andelements required for bone to heal itself. The physical characteristicsof cancellous bone make it an ideal material for a variety oforthopaedic applications including spinal fusions, maxillofacial andcraniofacial reconstruction, and treatment of long bone defects. Despitedesirable physical characteristics, cancellous bone may not be used insome applications because it is generally accepted that cancellousdemineralized bone has minimal or no osteoinductive capacity.

Bone is living, growing tissue. In vivo, bone is constantly beingrenewed. The old bone is removed and new bone is laid down. There aretwo phases, involving cellular activities, in this process. The firstphase, removal of old bone, is osteoclastic resorption. Osteoclastsdissolve some tissue on the surface of the bone and create a smallcavity. Typically this process takes place over a few days. The secondphase, laying down of new bone, is osteoblastic formation. Osteoblastsfill the cavities created by the osteoclasts with new bone. Typicallythis process takes a few months. Factors such as hormones, calcium,exercise, and other can affect the cells on the surface of bone andtrigger the remodeling cycle.

An osteoinductive bone matrix is provided by altering the naturalcollagenous structure of the bone. In some embodiments, theosteoinductive bone matrix is an osteoinductive cancellous bone matrix,thus providing a graft material with desirable physical characteristicsand osteoinductive capacity. In one embodiment, this is done by alteringthe collagenous structure of the bone such that the structure may be atleast partially resorbed. In another embodiment, this is done bycompacting the trabecular structure of the bone. In vivo, theosteoinductive bone matrix triggers the remodeling cycle. It is to beappreciate that, while the description herein may refer to cancellousbone for illustrative purposes, one skilled in the art would appreciatethat the discussion may be applied to cortical, corticocancellous, orcomposite bone.

II. Overview of Increasing the Osteoinductive Potential of Bone Matrix

Methods for increasing the biological activity of an at least partiallydemineralized bone matrix are provided. Osteoinductive osteoimplants arefurther provided. The osteoinductive osteoimplants comprise at leastpartially demineralized bone having increased biological activityrelative to at least partially demineralized bone that has not beenexposed to a treatment or condition as described herein. In someembodiments, the osteoinductive osteoimplants may comprise at leastpartially demineralized cancellous bone that has been exposed to atreatment or condition as described herein. The biological activitiesthat may be increased include but are not limited to osteoinductiveactivity, osteogenic activity, chondrogenic activity, triggering of boneremodeling, wound healing activity, neurogenic activity,contraction-inducing activity, mitosis-inducing activity,differentiation-inducing activity, chemotactic activity, angiogenic orvasculogenic activity, and exocytosis- or endocytosis-inducing activity.U.S. patent application Ser. No. ______ to Bone Matrix Compositions andMethods, filed Jun. 16, 2008, is herein incorporated by reference in itsentirety for the purposes of all that is disclosed therein. It will beappreciated that bone formation processes frequently include a firststage of cartilage formation that creates the basic shape of the bone,which then becomes mineralized (endochondral bone formation). Thus, inmany instances, chondrogenesis may be considered an early stage ofosteogenesis, though of course it may also occur in other contexts.

The at least partially demineralized bone, including wherein the atleast partially demineralized bone is at least partially demineralizedcancellous bone, provided herein exhibits osteoinductive activity. Theosteoinductive at least partially demineralized bone may be prepared byproviding at least partially demineralized bone and altering thecollagenous structure of the at least partially demineralized bone.Generally speaking, the osteoinductivity of the at least partiallydemineralized bone may be increased by either (1) altering the structureof the bone such that the trabecular density is increased while thecollagen structure is decreased, or (2) by opening up the structure ofthe bone, thus breaking the collagen apart and making growth factorswithin the bone more accessible. Thus, in certain embodiments, the atleast partially demineralized bone is exposed to a biological orchemical agent or to a combination of agents. The agent may be acleavage agent, e.g., a protease such as collagenase(s), or a chemicalagent such as cyanogen bromide. The matrix may be exposed to multipletreatments either together or sequentially. While not wishing to bebound by any theory, the treatment may alter the primary, secondary,tertiary, and/or quaternary structure of a component of the bone matrix(e.g., collagen, a bone morphogenetic protein, etc.) so as to increasethe biological activity of the matrix. An inventive treatment orcondition may “open up” the structure of the matrix, e.g., so as toallow biologically active molecules to be more readily released from ordiffuse within the matrix and/or to allow components such as nutrientsor growth-stimulatory molecules to enter the matrix. In certainembodiments the treatment or condition cleaves proteins present in theDBM (e.g., proteins such as bone morphogenetic proteins), which mayresult in conversion of an inactive protein into an active form, and/ormay generate an active molecule that is less susceptible to degradationthan a longer molecule from which it is derived. In certain embodiments,the bone is ground to a generally powder consistency and the powder isreaggregated into a dense structure through compression.

In certain embodiments, it is thought that the at least partiallydemineralized bone provided herein triggers the remodeling cycle.Specifically, remodeling of bone may be directed to sites that showdamage such as microcracks. It is theorized that the body recognizestissue regions that need repair and then sends cells to repair thoseregions. Thus, by treating or damaging the at least partiallydemineralized bone by, for example, treating or damaging the collagen,the at least partially demineralized bone is “marked” as requiringrepair. Thus, an implant formed from the cancellous at least partiallydemineralized bone is marked as requiring repair by cells. In someembodiments, inductive materials may be added to the implant to furtherstimulate bone remodeling. Alternatively, or additionally, nativeinductive materials in the bone may be exposed. The bone may becompacted to concentrate the inductive materials (added or native). Theimplant exhibiting tissue damage and, in some embodiments, increasedand/or concentrated inductive material triggers a rapid biologicresponse.

The treatment or condition may cleave an inhibitory factor that wouldotherwise inhibit a positively acting agent (an agent that enhances abiological activity of the bone matrix). For example, a variety ofproteins or protein fragments are known to inhibit the osteoinductiveand/or osteogenic activity of certain bone morphogenetic proteins, suchas BMP-2. In certain embodiments of the invention, the inhibitory effectof a protein or protein fragment may be reduced by exposing a bone orcartilage matrix to a treatment or condition. The treatment or conditionmay cause the cleavage or degradation of the inhibitory agent. Thetreatment or condition may block the interaction of the inhibitory agentwith its target (e.g., BMP-2) or may inhibit synthesis, secretion,post-translational modification, transport, etc. of the inhibitoryagent. For example, the bone matrix may be exposed to cleave antibodyinhibitory agents, or the antibody may be added to the bone matrix.

The osteoinductive at least partially demineralized bone may form anosteogenic and osteoinductive osteoimplant, discussed more fully below.In embodiments wherein the at least partially demineralized bone is atleast partially demineralized cancellous bone, the osteoimplant may havedesirable characteristics of cancellous bone and yet also haveosteoinductive capacity. The osteoimplant, when implanted in a mammalianbody, may induce, at the locus of the implant, the full developmentalcascade of endochondral bone formation including vascularization,mineralization, and bone marrow differentiation. Also, in someembodiments, the osteoinductive at least partially demineralized bonemay be used as a delivery device to administer bioactive agents.

Altering the Structure of the at Least Partially Demineralized Bone

In one embodiment, the collagenous structure of the at least partiallydemineralized bone is disrupted such that the demineralized bone may beat least partially resorbed after implantation. For example, at leastpartially demineralized bone may be altered so that it has a solidstructure at room temperature but substantially liquefies in the body orin tissue culture media such that it may have increased osteoinductiveactivity when compared to standard at least partially demineralizedbone.

Cancellous femoral bone has been shown to contain osteoinductive growthfactors that may be extracted from human bone. In one embodiment,cancellous DBM may be treated with, for example, collagenase, which isbelieved to open the structure of the cancellous DBM to make the growthfactors within the bone more bioaccessible. After treatment withcollagenase, the osteoinductive cancellous DBM may substantially liquefyin the body and may be at least partially resorbed by the body whenimplanted.

Compacting the Structure

Cortical bone includes dense lamellar structure. The structure ofcancellous bone is less dense than that of the lamellar structure ofcortical bone. If demineralized bone is considered to be a growth factorsource, the growth factor concentration gradients produced byimplantation of cancellous and cortical allograft may differsignificantly. By compressing the structure of cancellous bone, theosteoinductive potential may be increased. In some embodiments, thestructure of cortical bone may be compacted.

Compression of the at least partially demineralized bone may be achievedvia any suitable mechanism. For example, compression may be achieved bymechanical means, heat, chemical modification of the bone structure, anysuitable type of compression processing, or combinations of these. Inone embodiment, the bone is ground or otherwise processed into particlesof an appropriate size and formed into a dense bone structure. Grindingmay produce bone of a powder like consistency, the powder is wet, andthe wet bone powder is smeared to a consistency of fibrous paste. Thefibrous paste may be compressed and the paste may be permitted to dry.It is to be appreciated that the bone may be particulated before orafter demineralization. Thus, for example, the bone may be particulatedand thereafter partially demineralized. Alternatively, the bone may bedemineralized and then particulated.

In some embodiments, the bone may be compressed by loading cancellousbone in constrained compression, for example via palletizing the bone,without grinding the bone. Thus, in various embodiments, the at leastpartially demineralized bone is mechanically compacted into a densestructure. In yet other embodiments, the at least partiallydemineralized bone is compacted by treatment with chemicals, such aslithium chloride (LiCl), thereby shrinking the collagenous structure.

III. Providing Demineralized Bone

In some embodiments, demineralized bone that is substantially fullydemineralized is used. In other embodiments, partially demineralizedbone is used. In other embodiments, the surface demineralized bone isused. In other embodiments, nondemineralized bone may be used. In otherembodiments, combinations of some of all of the above may be used. Whilemany of the examples in this section refer to partially or surfacedemineralized bone, this is for illustrative purposes.

In one embodiment, the bone is partially demineralized. The at leastpartially demineralized bone may be provided in any suitable manner. Thebone useful in the invention herein is obtained utilizing methods wellknown in the art, e.g., allogenic donor bone. Bone-derived elements maybe readily obtained from donor bone by various suitable methods, e.g.,as described in U.S. Pat. No. 6,616,698, incorporated herein byreference in its entirety. The bone may be of autogenous, allogenic,xenogenic, or transgenic origin.

DBM preparations have been used for many years in orthopaedic medicineto promote the formation of bone. Typically, DBM preparations forpromoting the formation of bone have comprised cortical orcorticocancellous DBM. DBM has found use, for example, in the repair offractures, in the fusion of vertebrae, in joint replacement surgery, andin treating bone destruction due to underlying disease such asrheumatoid arthritis. Cortical DBM is thought to promote bone formationin vivo by osteoconductive and osteoinductive processes. Theosteoinductive effect of implanted cortical DBM compositions is thoughtto result from the presence of active growth factors present on theisolated collagen-based matrix. These factors include members of theTGF-β, IGF, and BMP protein families. Particular examples ofosteoinductive factors include TGF-β, IGF-1, IGF-2, BMP-2, BMP-7,parathyroid hormone (PTH), and angiogenic factors. Other osteoinductivefactors such as osteocalcin and osteopontin are also likely to bepresent in DBM preparations as well. There also are likely to be otherunnamed or undiscovered osteoinductive factors present in DBM.Cancellous DBM is understood to have little or no osteoinductivecapacity.

In one demineralization procedure of the present invention, the bone issubjected to an acid demineralization step and a defatting/disinfectingstep. The bone is immersed in acid over time to effect demineralization.Acids that may be employed in this step include inorganic acids such ashydrochloric acid and as well as organic acids such as formic acid,acetic acid, peracetic acid, citric acid, propionic acid, etc.Demineralization may be to a point where the bone is fullydemineralized, partially demineralized, or surface demineralized. Thedepth of demineralization into the bone surface may be controlled byadjusting the treatment time, temperature of the demineralizingsolution, concentration of the demineralizing solution, and agitationintensity during treatment.

The at least partially demineralized bone is rinsed with sterile waterand/or buffered solution(s) to remove residual amounts of acid andthereby raise the pH. A suitable defatting/disinfectant solution is anaqueous solution of ethanol, the ethanol being a good solvent for lipidsand the water being a good hydrophilic carrier to enable the solution topenetrate more deeply into the bone particles. The aqueous ethanolsolution also disinfects the bone by killing vegetative microorganismsand viruses. Ordinarily, at least about 10 to 40 percent by weight ofwater (i.e., about 60 to 90 weight percent of defatting agent such asalcohol) should be present in the defatting disinfecting solution toproduce optimal lipid removal and disinfection within the shortestperiod of time. In a particular embodiment, the concentration range ofthe defatting solution is from about 60 to about 85 weight percentalcohol. In a further embodiment, the defatting solution is about 70weight percent alcohol. In yet another embodiment, the cancellous boneis defatted in a solution of 1:1 chloroform:methanol at room temperatureand then demineralized in 0.6 N HCl at 4° C.

The at least partially demineralized bone may be ground or otherwiseprocessed into particles of an appropriate size before or afterdemineralization. In certain embodiments, the particle size is greaterthan 75 microns, ranges from about 100 to about 3000 microns, or rangesfrom about 100 to about 800 microns. After grinding the bone to thedesired size, the mixture may be sieved to select those particles of adesired size. In certain embodiments, the bone particles may be sievedthough a 50 micron sieve, a 75 micron sieve, or a 100 micron sieve.

Generally, demineralization conditions may affect the osteoinductivityof DBM. Proteases that degrade the osteoinductive activity of corticalbone have been described. Proteases present in cancellous bone have beenshown to differ from those responsible for cortical bone remodeling.Thus, demineralization conditions utilized for cortical bone processingmay not be ideal for cancellous bone. Specific protease inhibitorcocktails and lower temperatures may be used.

Following particulation, the bone may be treated to remove mineral fromthe bone as discussed above. While hydrochloric acid is commonly used asa demineralization agent, there are other methods for preparing DBM,which vary widely and include choices regarding the concentration of thedemineralization agent; the temperature and duration of thedemineralization step; the inclusion or exclusion at various points inthe demineralization process of solvents or solvent combinations such asethanol, methanol, and chloroform:ether; the extent to which the matrixis washed following the demineralization step; and whether the resultingDBM is stored frozen or is lyophilized and stored at room temperature.See, for example, Russell et al., 22(5) Orthopaedics 524-531 (1999),incorporated herein by reference.

Any of a variety of at least partially demineralized bone preparationsmay be used with the present invention. DBM prepared by any method maybe employed, including particulate or fiber-based preparations, mixturesof fiber and particulate preparations, fully or partially demineralizedpreparations, mixtures of fully and partially demineralizedpreparations, and surface demineralized preparations. See U.S. Pat. No.6,326,018; Reddi et al., 69 Proc. Natl. Acad. Sci. USA 1601-1605 (1972);Lewandrowski et al., 317 Clin. Ortho. Rel. Res. 254-262 (1995);Lewandroski et al., 31 J. Biomed. Mater. Res. 365-372 (1996);Lewandrowski et al. 61 Calcified Tiss. Int., 294-297 (1997);Lewandrowski et al., 15 I Ortho. Res. 748-756 (1997), each of which areincorporated herein by reference in their entireties. Suitabledemineralized bone matrix compositions are described in U.S. Pat. No.5,507,813, hereby incorporated by reference in its entirety. In someinstances, large fragments or even whole bone may be demineralized, andthen particulated following demineralization.

In some embodiments, the bone may be surface demineralized. The surfacemay be an inner surface, such as inside trabeculae or inside aHalversion canal. In other embodiments the surface may be an outersurface. In some embodiments, surface demineralized refers to the bonecomprising at least one outer surface, or zone of an outer surface, thatis demineralized and possessing a non-demineralized core. In someembodiments, the entirety of the surface may be partially demineralized.In other embodiments, a portion of the surface may be demineralized,such as by exposing only a portion of a particle to the demineralizationprocess, by exposing a portion of the surface to a greater or lesserextent of the demineralization process, by masking, etc.Demineralization may be done to a certain percentage. In someembodiments, that percentage relates to weight percentage. In otherembodiments, that percentage relates to percentage of the size of thebone being demineralized, or to the depth of demineralization. The depthof demineralization of the at least one outer surface thus may be viewedas a percentage of the size of the bone being demineralized or may beviewed as an absolute number.

Demineralization thus may be carried out to a percentage depth of thesize of the bone being demineralized. FIGS. 4 a and 4 b illustratesurface demineralized bone particles. The bone particle 100 of FIG. 2 ais substantially spherical. The bone particle 110 of FIG. 4 b issomewhat elongate.

As shown, the bone particle 100 of FIG. 2 a has a demineralized surfaceregion 106 and a non-demineralized core 108. The bone particle 100includes a length 102 along its longest dimension and a length 104 alongits shortest dimension. The length 102 in the longest dimensioncomprises first and second demineralized portions 103 a and 103 b and anondemineralized portion 105. A percentage of demineralization in thelongest dimension may be determined by summing the length of the firstand second demineralized portions 103 a and 103 b and dividing thattotal by the length 102 (comprising 103 a, 103 b and 105). The length104 in the shortest dimension likewise comprises first and seconddemineralized portions 107 a and 107 b and a nondemineralized portion109. A percentage of demineralization in the shortest dimension may bedetermined by summing the length of the first and second demineralizedportions 107 a and 107 b and dividing that total by the length 104(comprising 107 a, 107 b and 109). A total percentage demineralizationmay be determined by averaging the percent demineralization in thelongest dimension with the percent demineralization in the shortestdimension.

As shown, the bone particle 110 of FIG. 2 b has a demineralized surfaceregion 116 and a non-demineralized core 118. The bone particle 110includes a length 112 along its longest dimension and a length 114 alongits shortest dimension. The longest dimension and shortest dimension aretaken as those measuring largest and smallest, respectively, such as bya micrometer or using other by suitable manner and generally goingthrough the center of the bone particle 110. The length 112 in thelongest dimension comprises first and second demineralized portions 113a and 113 b and a nondemineralized portion 115. A percentage ofdemineralization in the longest dimension may be determined by summingthe length of the first and second demineralized portions 113 a and 113b and dividing that total by the length 112 (comprising 113 a, 113 b,and 115). The length 114 in the shortest dimension likewise comprisesfirst and second demineralized portions 117 a and 117 b and anondemineralized portion 119. A percentage of demineralization in theshortest dimension may be determined by summing the length of the firstand second demineralized portions 117 a and 117 b and dividing thattotal by the length 114 (comprising 117 a, 117 b, and 119). A totalpercentage demineralization may be determined by averaging the percentdemineralization in the longest dimension with the percentdemineralization in the shortest dimension.

Alternatively, percentage demineralization may be based on weightpercent demineralized of total weight of the bone particle.

In some embodiments, demineralization may be carried out to a depth of,for example, at least about 100 microns. Surface demineralization mayalternatively be done to a depth less than or more than about 100microns. Generally, surface demineralization may be done to a depth ofat least 50 microns, at least 100 microns, at least 200 microns, orother. Accordingly, in some embodiments, the demineralized bonecomprises at least one outer surface possessing at least onedemineralized zone and a non-demineralized core, wherein thedemineralized zone of the outer surface of the bone may be, for example,at least about 100 microns thick. The demineralized zone mayalternatively be less than or more than about 100 microns thick. Thedemineralized zone of the surface of the bone is osteoinductive, andtherefore promotes rapid new ingrowth of native host bone tissue into anosteoimplant comprising surface demineralized bone. The osteoimplant maycomprise surface demineralized monolithic bone or an aggregate ofsurface demineralized bone particles, and may be substantially solid,flowable, or moldable. The demineralized zone of the surface of the bonecan be any surface portion.

When it is desirable to provide an osteoimplant having improvedbiological properties while still substantially maintaining the strengthpresent in the osteoimplant prior to demineralization, for example wheremonolithic bone is used, the extent and regions of demineralization ofthe monolithic bone may be controlled. For example, depth ofdemineralization may range from at least about 100 microns to up toabout 7000 microns or more, depending on the intended application andgraft site. In some embodiments, the depth of demineralization isbetween 100 to about 5000 microns, between about 150 to about 2000microns, or between about 200 microns to about 1000 microns. Inalternative embodiments, depth of demineralization may be less thanabout 100 microns. Reference is made to U.S. Pat. No. 7,179,299, hereinincorporated by reference for discussion of surface demineralization.

A benefit of surface demineralized bone is that the demineralizedzone(s) can elastically yield under applied force while the mineralizedcore has strength and load bearing capacity exceeding that ofdemineralized bone. Thus, when the surface demineralized bone issubjected to an applied load, the demineralized zones can conform tocontours of adjacent bone tissue and thereby minimize voids or spacesbetween the osteoimplant and adjacent bone tissue. This can be usefulbecause host bone tissue will not grow to bridge large voids or spaces.Thus, by conforming to the contours of adjacent bone tissue, anosteoimplant comprising surface demineralized monolithic bone exhibitsenhanced biological properties such as, for example, incorporation andremodeling. The non-demineralized inner core imparts mechanical strengthand allows the monolithic osteoimplant to bear loads in vivo. Othernon-demineralized zones provide improved tolerances when engaged withother objects such as, for example, insertion instruments, otherimplants or implant devices, etc. It is noted that some of thesecharacteristics may also be exhibited by an osteoimplant comprising anaggregate of surface-demineralized bone particles.

IV. Enhancing Osteoinductive Properties

In certain embodiments, the treatment or condition alters a biologicalactivity of the at least partially demineralized bone such that the atleast partially demineralized bone displays osteoinductive, osteogenic,and/or chondrogenic activity in a species in which a control matrix(e.g., an inactivated matrix or a matrix not exposed to the treatment orcondition) does not show such activity, or shows it in a lesser amount.For example, at least partially demineralized bone exposed to thetreatment or condition may display increased osteoinductive, osteogenic,and/or chondrogenic activity in human, dog, squirrel monkey, etc., asassessed either in vitro or in vivo. Specifically, in some embodiments,cancellous bone, which is understood to have little to noosteoinductivity, as treated herein displays osteoinductive capacity.

Altering the Structure of the at Least Partially Demineralized Bone

In one embodiment, the structure of the at least partially demineralizedbone may be altered to make growth factors within the bone moreaccessible. Thus, the at least partially demineralized bone may becontacted with a cleavage agent, e.g., a protease such as collagenase(s)or a chemical agent such as cyanogen bromide. The cleavage agents may beapplied either together or sequentially, optionally washing the matrixbetween application of different agents to remove residual agent. Ingeneral, the biological and chemical agents may be used in an effectiveamount and for a time sufficient to achieve a desired outcome, e.g., adesired increase in a biological activity of the matrix.

In one embodiment, treatment may be by limited digestion with purifiedbacterial collagenase. Collagenases and their activity on collagens ofvarious types have been extensively studied. A number of collagenasepreparations are available from Worthington Biochemical Corporation, ofLakewood, N.J. As is known in the art, collagen consists of fibrilscomposed of laterally aggregated, polarized tropocollagen molecules (MW300,000). Each tropocollagen unit consists of three helically woundpolypeptide α-chains around a single axis. The strands have repetitiveglycine residues at every third position and numerous proline andhydroxyproline residues, with the particular amino acid sequence beingcharacteristic of the tissue of origin. Tropocollagen units combineuniformly to create an axially repeating periodicity. Cross linkagescontinue to develop and collagen becomes progressively more insolubleand resistant to lysis on aging. Gelatin results when solubletropocollagen is denatured, for example on mild heating, and thepolypeptide chains become randomly dispersed. In this state the strandsreadily may be cleaved by a wide variety of proteases.

In the past, digestion of bone with purified bacterial collagenase hasbeen used for altering the solubility of human DBM in cell culturemedia. Digestion of bone with purified bacterial collagenase has alsobeen used to enhance the response of myoblastic cells to cortical humanDBM.

As taught herein, cancellous DBM treated with collagenase has enhancedin vivo osteoinductive capacity. Collagenase treatment of cancelloushuman DBM increases its solubility relative to that of untreatedcancellous human DBM. The solubility of the cancellous DBM may beincreased by exposure to an appropriate treatment or condition, e.g.,collagenase treatment, radiation, heat, or any other suitable treatmentand/or condition, etc. The extent to which the solubility is increasedmay be varied by varying the nature of the treatment (e.g., the enzymeconcentration) and/or the time over which it is applied. In someimplementations, the cancellous DBM may be partially solubilized by thecollagenase, while in others, the cancellous DBM may be completelysolubilized by the collagenase.

According to various embodiments, a combination of treatments may beused. For example, the partially digested at least partiallydemineralized bone may be treated with heat or pepsin or anotherprotease to disrupt cross-links not disrupted by the collagenase. The atleast partially demineralized bone may be exposed to a variety ofbiological agents in addition to, or instead of, one or more proteases.Other enzymes include methylases, acylases, lipases, phospholipases,endo- and exo-glycosidases, glycanases, glycolases, amylase, pectinases,galacatosidases, etc. Chemical agents that perform similar reactions maybe used. For example, a number of different alkylating agents are known.A variety of salts present in high concentrations (e.g., at least 6 M,7M, 8M, etc.) may be used. Exemplary salts include salts of variousGroup I elements, e.g., LiCl. Denaturing agents, e.g., denaturing saltssuch as guanidinium HCl, may be used. Where denaturing agents are used,care should be taken to avoid denaturing desired components present inthe matrix, e.g., growth factors.

A variety of different collagenases known in the art may be used.Collagenases are classified in section 3.4.24 under the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB) enzymenomenclature recommendations (see, e.g., 3.4.24.3, 3.4.24.7, 3.4.24.19).The collagenase may be of eukaryotic (mammalian) or prokaryotic(bacterial) origin. Bacterial enzymes differ from mammalian collagenasesin that they attack many sites along the helix. Collagenase may cleavesimultaneously across all three chains or attack a single strand.Generally, the collagenase cleaves Type I collagen, e.g., degrades thehelical regions in native collagen, at the Y-Gly bond in the sequencePro-Y-Gly-Pro-, where Y is most frequently a neutral amino acid. Thiscleavage yields products susceptible to further peptidase digestion. Anyprotease having one or more of these activities associated withcollagenase may be used as a collagenase in accordance with the presentinvention.

It will be appreciated that crude collagenase preparations contain notonly several collagenases, but also a sulfhydryl protease, clostripain,a trypsin-like enzyme, and an aminopeptidase. This combination ofcollagenolytic and proteolytic activities is effective at breaking downintercellular matrices, the essential part of tissue disassociation.Crude collagenase is inhibited by metal chelating agents such ascysteine, EDTA, or o-phenanthroline, but not DFP. It is also inhibitedby α2-macroglobulin, a large plasma glycoprotein. Ca²⁺ is required forenzyme activity. Therefore, it may be desirable to avoid collagenaseinhibiting agents when treating at least partially demineralized bonewith collagenase. In addition, although the additional proteases presentin some collagenase preparations may aid in breaking down tissue, theyalso may cause degradation of desired matrix constituents such as growthfactors. Therefore, it may be desirable to use a highly purifiedcollagenase that contains minimal secondary proteolytic activities alongwith high collagenase activity. For example, a collagenase preparationmay contain at least 90%, at least 95%, at least 98%, or at least 99%collagenase by weight. The preparation may be essentially free ofbacterial components, particularly bacterial components that could causeinflammatory or immunological reactions in a host, such as endotoxin,lipopolysaccharide, etc. Preparations having a purity greater than 99.5%may be used. It may be desirable to include various protease inhibitorsthat do not inhibit collagenase but that inhibit various proteases thatdigest BMP. For example, protease inhibitors that are known to protectBMP activity from degradation include N-ethyl maleimide, benzamidinehydrochloride, iodoacetic acid, PMSF, AEBSF, and E-64. Bestatin may alsobe used, particularly if the preparation contains aminopeptidaseactivity. Any of these protease inhibitors (or others) may be includedin a carrier, such as a bone matrix composition, or in any compositionthat is used to treat a carrier.

As discussed above, collagenase disrupts crosslinks between collagen.Using highly purified collagenase, it is possible that not all thecrosslinks are disrupted. Generally, different grades of collagenase maydisrupt different ranges of crosslinks. Thus, other treatments, such aspepsin treatment or heat treatment, may be used to disrupt crosslinksnot affected by the collagenase.

In alternative embodiments, any suitable compound for altering thestructure of the DBM may be used. For example, enzymes (e.g., pepsin) orchemicals may be used. Pepsin alters the structure of Type I collagen bycleaving the associated telopeptides. Further, mechanical means such asionizing radiation or electromagnetic radiation may be used.

Another suitable protease is bone morphogenetic protein 1 (BMP-1). BMP-1is a collagenolytic protein that has also been shown to cleave chordin(an inhibitor of BMP-2 and BMP-4). Thus, BMP-1 may be of use to alterthe physical structure of the DBM (e.g., by breaking down collagen)and/or to cleave specific inhibitory protein(s), e.g., chordin ornoggin. Proteins related to any of the proteases described herein, i.e.,proteins or protein fragments having the same cleavage specificity, alsomay be used. It will be appreciated that variants having substantialsequence identity to naturally occurring protease may be used. Forexample, variants at least 80% identical over at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100% of the length ofnaturally occurring protease (or any known active fragment thereof thatretains cleavage specificity) when aligned for maximum identity allowinggaps may be used.

Certain proteases that may provide desirable effects include members ofthe proprotein convertase (PPC) family of proteases, such as furin andrelated proteases. Members of this family of cellular enzymes cleavemost prohormones and neuropeptide precursors. Numerous other cellularproteins, some viral proteins, and bacterial toxins that are transportedby the constitutive secretory pathway are also targeted for maturationby PCs. Furin and other PC family members share structural similaritiesthat include a heterogeneous ˜10 kDa amino-terminal proregion, a highlyconserved ˜55 kDa subtilisin-like catalytic domain, andcarboxyl-terminal domain that is heterogeneous in length and sequence.These enzymes become catalytically active following proregion cleavagewithin the appropriate cellular compartment. Furin is the majorprocessing enzyme of the secretory pathway and is localized in thetrans-golgi network. van den Ouweland, A. M. W. et al., 18 Nucl. AcidRes. 664 (1990); Steiner, D. F., 2 Curr. Opin. Chem. Biol. 31-39 (1998).Substrates of furin include blood clotting factors, serum proteins, andgrowth factor receptors such as the insulin-like growth factor receptor.Bravo D. A. et al., 269 J. Biol. Chem. 25830-25873 (1994). The minimalcleavage site for furin is Arg-X-X-Arg. However, the enzyme prefers thesite Arg-X-(Lys/Arg)-Arg. An additional arginine at the P6 positionappears to enhance cleavage. Krysan D. J. et al., 274 J. Biol. Chem.23229-23234 (1999). Furin is inhibited by EGTA, α1-antitrypsin Portland,Jean, F. et al., 95 Proc. Natl. Acad. Sci. USA 7293-7298 (1998), andpolyarginine compounds, Cameron, A. et al., 275 J. Biol. Chem.36741-36749 (2000). Furin has been shown to proteolytically process bothproTGF and proBMP proteins, for example, proTGF-β and proBMP-4,respectively, resulting in the release of the active mature form foreach molecule. Dubois et al., 158(1) Am. J. of Pathology 305-316 (2001);Cui et al., 17(16) Embo Journal 4735-4743 (1998); Cui et al., 15 Genes &Development 2797-2802 (2001), each incorporated by reference herein intheir entireties. Furin has also been shown to cleave BMP-2, BMP-6, andBMP-7. For example, furin cleaves between amino acids 282 and 283 inmature human BMP-2. Newly synthesized human BMP-2 contains a signalsequence (amino acids 1-23), a propeptide (amino acids 24-282), and anactive portion (amino acids 283-396). Furin cleaves mature BMP-2 (aminoacids 24-396) between amino acids 282 and 283 to release the propeptideand the active molecule.

Thus, the at least partially demineralized bone may be treated with PPCssuch as furin and/or other proteases, which process immature TGF-βand/or BMP superfamily propeptides into their active mature forms and/orprocess active or inactive TGF-β and/or BMP superfamily polypeptidesinto smaller active fragments that are resistant to degradation orinactivation relative to the longer polypeptide, generating at leastpartially demineralized bone with increased osteoinductivity compared toat least partially demineralized bone lacking the protease, resulting inimproved bone formation. The higher titers of the mature and/ordegradation resistant species in these preparations increase theosteoinductive capacity of the at least partially demineralized bone.

At least partially demineralized bone may be exposed to any of theenzymes, e.g., proteases described herein (and others) at a range ofdifferent concentrations, e.g., between 1 pg/ml-100 μg/ml. For example,a protease may be used at between 1 pg/ml-100 pg/ml, between 100 pg/mland 1 ng/ml, between 1 ng/ml and 100 ng/ml, between 100 ng/ml and 1μg/ml, between 1 μg/ml and 100 μg/ml, etc. A variety of differentdigestion buffers may be used. The time of digestion may vary accordingto the protease, amount of DBM, and desired degree of digestion. Ingeneral, suitable times range between 30 minutes to 72 hours, e.g.,between 30 minutes to 1 hour, between 1 and 12 hours, between 12 and 24hours, between 24 and 48 hours, between 48 and 72 hours, etc. It will beappreciated that these times are approximate. Determination of theoptimal treatment times for any preparation may involve assay of thetreated tissue preparation in one of the biological activity assaysdescribed herein or others known in the art.

Allogenic cancellous demineralized bone is known not to beosteoinductive. Schwarz et al., 111(1) Arch Orthop Trauma Surg. 47-50(1991). Applicants have made the surprising discovery that allogeniccancellous bone, when treated with collagenase enzymes for approximatelyone hour, becomes osteoinductive, approximately as osteoinductive asallogenic cortical demineralized bone. Thus, the bone, whethercancellous, corticocancellous, or cortical, may be treated for anysuitable time period to enhance its osteoinductivity, including fromabout 15 minute to about 3 hours, from about 30 minutes to about 90minutes, or for about 60 minutes. While not desiring to be bound by anyparticular scientific theory, applicants state that it is believed thatthe disruption of the collagen matrix makes osteoinductive factorsbioavailable. Many types of collagenalytic enzymes, such as those setforth herein, would be expected to render allogenic at least partiallydemineralized bone osteoinductive when treated as described herein.Other treatments alternatively may be used and also are expected toprovide the same result, including the use of salts or ionizing orelectromagnetic radiation, or various categories of enzymes, so long asthe enzymes disrupt the collagen without damaging the osteoinductivefactors.

In addition, the osteoinductive capacity of DBM preparations in higherorder species, such as squirrel monkeys, dogs, and humans, has beenquestioned. For example, allogeneic cancellous bone blocks,demineralized or not, have no osteoinductive capacity and noosteoconductive function that promotes healing of mid-diaphyseal bonedefects in dogs. Schwarz et al., 111(1) Arch. Orthop. Trauma Surg. 47-50(1991). Adult monkey bone matrix contains bone inductive properties, butthese properties are not sufficient to induce bone formation in adultmonkey muscle sites. Aspenberg et al., 63(6) Acta Orthop Scand. 619-622(1992); see also Aspenberg et al., 9(1) J. Orthop. Res. 20-5 (1991);Schmid et al., 19(1) Unfallchirurgie 1-8 (1993); Toriumi et al., 116(6)Arch Otolaryngol Head Neck Surg. 676-680 (1990); and Ousterhout, D K,15(5) Ann. Plast Surg. 367-373 (1985). Structural modifications ofdemineralized bone collagen may serve to activate the latentosteoinductive potential of DBM preparations in higher order species.Such products, irrespective of whether derived from cancellous orcortical bone, may have significantly enhanced clinical efficacy in avariety of orthopaedic applications as observed by more rapid bonehealing and spinal fusion.

The at least partially demineralized bone may be exposed to a physicalcondition instead of, or in addition to, a biological or chemical agent.For example, the at least partially demineralized bone may be exposed toheat or cold for a suitable period of time, e.g., minutes, hours, or upto several days, where “heat” refers to temperatures above roomtemperature (about 23-25 degrees C.) and “cold” refers to temperaturesbelow room temperature. Cycles of temperature change may be used, e.g.,the at least partially demineralized bone may be heated and cooled aplurality of times. The temperature may, for example, be at least 37degrees C., at least 40, 50, 60, 70, 80, or 90 degrees C. In someembodiments, the heat treatment may be relatively gentle to avoiddenaturing growth factors and other factors, typically proteins orpeptides, that contribute to the osteogenic, osteoinductive, orchondrogenic activity of the matrix. The temperature may be 20 degreesC. or below, 15 degrees C. or below, 10 degrees C. or below, 0 degreesC. or below, etc. In general, the at least partially demineralized bonemay be exposed to any desired temperature in the presence or absence ofother agents, solvents, or other suitable media. The at least partiallydemineralized bone may be exposed to electromagnetic energy of any type,e.g., X-rays and microwaves. Gamma-rays, beta-rays, or any suitableionizing radiation may be used. The treatment may be performed in theabsence of oxygen or in a reduced oxygen environment. U.S. patentapplication Ser. No. ______ to Method of Treating Tissue, filed Jun. 16,2008, is herein incorporated by reference in its entirety for thepurposes of all that is disclosed therein.

In certain embodiments, the treatment or condition alters the physicalstructure of the matrix so as to increase its biological activity. Thetreatment or condition may alter the structure of the at least partiallydemineralized bone so as to facilitate the presentation of suchmolecules, e.g., on a surface of the at least partially demineralizedbone. The treatment or condition may alter the conformation of suchmolecules in a manner that facilitates interactions with target cells,e.g., cells that migrate towards or into the bone matrix. The treatmentor condition may alter release kinetics of agents such as growthfactors, differentiation factors, chemotactic factors, etc., from thematrix. Exemplary factors that up-regulate collagen synthesis byosteoblasts include TGF-β, PDGF, IGF, IL-1, PGE₂, and certain BMPs.Certain treatments may alter, e.g., increase, the affinity of boneand/or cartilage forming cells and/or undifferentiated cells capable ofdifferentiation into bone and/or cartilage forming cells for the matrix.For example, the treatment or condition may alter integrin binding sites(such as RGD sequences), e.g., by making them more available to cells.

In certain embodiments, alteration of the structure involves cleavage orpartial degradation of one or more major structural component of thematrix such as collagen, e.g., components that typically make up atleast 1%, 5%, 10%, 25%, 50%, 75%, 90% etc., of the dry weight of the atleast partially demineralized bone. In certain embodiments, thesecondary, tertiary, and/or quaternary structure of a major structuralcomponent of the matrix is altered. The alteration may includedestruction of bonds that normally maintain the triple helical structureof collagen, bonds that hold collagen fibrils together, etc. DBM is adense structure held together by cross-linked collagen. Most of thenoncollagenous proteins (NCPs) are trapped within and/or attached tothis framework. Certain agents such as collagenase may cut across theframework and thereby potentially allow access to the NCPs. The amountof collagen (or other structural protein) that is cleaved and/ordegraded may vary. For example, in certain embodiments, at least 10%, atleast 25%, at least 50%, at least 75%, or at least 90% of the collagenoriginally present in the DBM is cleaved or degraded. Between 10-25%,25-50%, 50-75%, 75-90%, 90-100%, or any other suitable range such as10-90%, 25-75%, etc., of the collagen may be cleaved or degraded. Apolypeptide is considered to be cleaved if it is cleaved at a singlesite or at multiple sites. In certain embodiments, the cleavage cleavesa crosslink. In certain embodiments, at least a portion of the collagenis present as collagen fragments. For example, at least 10%, at least25%, at least 50%, at least 75%, at least 90%, etc., of the collagen ispresent as collagen fragments in certain embodiments. Between 10-25%,25-50%, 50-75%, 75-90%, 90-100%, or any other range such as 10-90%,25-75%, etc., of the collagen may be present as collagen fragments. Thefragments may remain associated with or present in or on the bone matrixor may diffuse away. A bone matrix may be exposed to any of a variety ofdifferent biological or chemical agents or conditions for different timeperiods in order to achieve a desired degree of cleavage or degradationof a structural component of the matrix such as collagen. The inventiontherefore provides a modified bone matrix comprising acollagen-containing bone matrix, wherein at least a portion of thecollagen is cleaved or degraded. Matrices in which at least a portion ofa different structural component of the matrix is cleaved or degradedare also provided.

In certain embodiments, the at least partially demineralized bone may beexposed to a treatment or condition that generates peptides and proteinfragments having osteoinductive or chondrogenic activity. In contrast tovarious longer proteins, certain peptides and protein fragments are lesssusceptible to proteolytic degradation and more likely to maintain theirosteoinductive or chondrogenic properties in the proteolytic environmentof the matrix or implant site. Many osteoinductive and chondrogenicproteins, for example, growth factors such as BMPs, cell signalingmolecules, transcription factors, hormones, etc., have domains that areresponsible for binding to receptors and/or initiating signaltransduction in bone and cartilage growth pathways. These domains arecapable of functioning independently as peptides and protein fragments.In certain embodiments, the present invention increases theosteoinductive or chondrogenic activity of bone and cartilage matricesby cleaving the osteoinductive and chondrogenic factors present in thematrix to generate active peptides or protein fragments and/or togenerate active peptides or protein fragments that are less susceptibleto degradation than their longer precursors. The increased number offactors in the matrix results in increased bone or cartilage formation.

Compacting the Structure

The at least partially demineralized bone may be compacted to increasethe growth factor concentration gradient of the at least partiallydemineralized bone. Thus, by compressing the structure of bone, theosteoinductive potential may be increased. Compression may be achievedvia any suitable mechanism. For example, compression may be achieved bymechanical means, heat, chemical modification of the collagenousstructure or any suitable type of compression processing. In oneembodiment, the bone is compacted by grinding or otherwise processingthe bone into particles of an appropriate size and formed into a densebone structure. In another embodiment, the bone is compacted bytreatment with LiCl, thereby shrinking the structure.

Cancellous DBM has a sponge-like structure. Accordingly, in someembodiments, use of compression may be particularly suitable forcancellous bone. While the DBM may be compressed, typically whencontacted with a liquid the DBM returns to its pre-compressed state. Toprevent the DBM from returning to its pre-compressed state, the DBM maybe provided in a container for maintaining force on it. Thus, forexample, it may be placed in a defect, mesh, chamber, cage, etc. Inaccordance with some embodiments, the pressed DBM structure may bespecifically configured to retain its shape in water for a predeterminedamount of time. For example, the pressed DBM structure may be configuredto retain its shape in water for one or more hours, up to seven hours,up to several days, e.g., 48, 72 or 96 hours, or up to one week orseveral weeks.

The osteoinductive at least partially demineralized bone may be used inan expandable osteoimplant such as taught by PCT Application No.PCT/US2006/001540, filed Jan. 17, 2006, hereby incorporated by referencein its entirety. As disclosed in that application, an osteoimplantcomprising demineralized bone particles has a first state and anexpanded state. The osteoimplant may be used with another device or onits own. In the first state, the osteoimplant may be inserted into adevice such as an intervertebral body fusion device. The osteoimplantmay be rehydrated to expand to an increased size, for example as far aspermitted by the confines of the intervertebral body fusion device andspinal endplates, thereby aiding in greater vertebral endplate contactand conformity in spinal surgery. In addition or alternatively,rehydration may take place in vivo. The osteoimplant may comprise fullyor partially demineralized cancellous bone and fully and/or partiallydemineralized cortical bone. In one embodiment, the cancellous bone isprovided in chips, and the cortical bone in fibers. The demineralizedcancellous bone may comprise, in whole or part, osteoinductivecancellous DBM as taught herein. In another embodiment, the expandableosteoimplant may comprise, in whole or in part, cancellous DBM, some orall of which may be made osteoinductive pursuant to the presentinvention. In yet a further embodiment, the expandable osteoimplant maycomprise a monolithic piece of bone treated as provided herein, such asby at least partially demineralizing and treating the monolithic pieceof bone.

In one embodiment, the compressed at least partially demineralized boneis provided by grinding the bone, for example into a fine powder, andmolding it into a cohesive shape. The bone may be wetted and processedto a consistency of a fibrous paste before molding. In one embodiment,the molding is done via hand molding and drying the material at ambienttemperature. In another embodiment, the molding is done by putting thebone in a mold, such as a polyurethane or PTFE mold, putting the moldunder pressure, for example by clamping the mold, and allowing the boneto dry, for example via vacuum drying the material at 40° C. for 72hours. In yet another embodiment, the molding is done by putting thebone into a stainless steel mold, pressing the mold with 500 pounds andvacuum drying the mold at 40° C. for 72 hours with weighteddehydrothermal plungers.

Thus, in various embodiments, the DBM may be ground to a powder-likeconsistency, processed into a fibrous paste, and the fibrous pastecompressed and dried. Compression and drying may be done in any suitablemanner. For example, compression may be done by applying manualpressure, by clamping (powered manually, through dehydrothermalplungers, or other), or other means. Drying may be done in air atambient temperature, in an oven, vacuum dried, or in any other suitablemanner.

In another embodiment, the at least partially demineralized bone iscompacted by treatment with LiCl to shrink the collagenous structure.Specifically, at least partially demineralized bone may be treated with8M LiCl to increase the osteoinductivity of the at least partiallydemineralized bone. Treatment with LiCl may further increase theradioopacity of the at least partially demineralized bone.

V. Osteoimplant

Bone grafting applications are differentiated by the requirements of theskeletal site. Certain applications generally use a “structural graft”in which one role of the graft is to provide mechanical or structuralsupport to the site. Such grafts contain a substantial portion ofmineralized bone tissue to provide the strength needed for load-bearing.Examples of applications requiring a “structural graft” includeintercalary grafts, spinal fusion, joint plateaus, joint fusions, largebone reconstructions, etc. Other applications generally use an“osteogenic graft,” in which one role of the graft is to enhance oraccelerate the growth of new bone tissue at the site. Such graftscontain a substantial portion of demineralized bone tissue to improvethe osteoinductivity needed for growth of new bone tissue. Examples ofapplications requiring “osteogenic graft” include deficit filling,spinal fusions, joint fusions, etc. Grafts may also have otherbeneficial biological properties, such as, for example, serving asdelivery vehicles for bioactive substances. Bioactive substances includephysiologically or pharmacologically active substances that act locallyor systemically in the host.

In accordance with various embodiments, the at least partiallydemineralized bone matrix provided herein may be used as a structuralgraft, an osteogenic graft, or a graft suitable for both structural andosteogenic uses.

Any suitable shape, size, and porosity of at least partiallydemineralized bone may be used. In various embodiments, the bone may bemonolithic or may be composite. Rat studies show that the new bone isformed essentially having the dimensions of the device implanted. Asuccessful osteoimplant for encouraging bone development appropriatelyaccommodates each step of the cellular response during bone development,and in some cases, protects osteoinductive factors from nonspecificproteolysis. In some uses, the osteoimplant acts as a temporary scaffolduntil replaced completely by new bone. In bone, the dissolution ratesmay vary according to whether the implant is placed in cortical ortrabecular bone.

The osteoimplant resulting from the at least partially demineralizedbone may assume a determined or regular form or configuration such as asheet, plate, disk, tunnel, cone, tube, a rod, string, weave, solid,fiber, or wedge, to name but a few. Prefabricated geometries include,but are not limited to, a crescent apron for single site use, an I-shapeto be placed between teeth for intra-bony defects, a rectangular bib fordefects involving both the buccal and lingual alveolar ridges,neutralization plates, reconstructive plates, buttress plates,T-buttress plates, spoon plates, clover leaf plates, condylar plates,compression plates, bridge plates, wave plates. Partial tubular as wellas flat plates may be fabricated from the osteoimplant. Such plates mayinclude such conformations as, e.g., concave contoured, bowl shaped, ordefect shaped.

In certain embodiments, the shape and size of the particles in thecarrier affect the time course of osteoinductivity. For example, in acone or wedge shape, the tapered end will result in osteoinductivityshortly after implantation of the osteoimplant, whereas the thicker endwill lead to osteoinductivity later in the healing process (hours todays to weeks later). Also, larger particle size will have induce boneformation over a longer time course than smaller particles. Particles ofdifferent characteristics (e.g., composition, size, shape) may be usedin the formation of these different shapes and configurations. Forexample, in a sheet of DBM a layer of long half-life particles may bealternated between layers of shorter half-life particles. See U.S. Pat.No. 5,899,939, herein incorporated by reference in its entirety, forsuitable examples. In a weave, strands composed of short half-lifeparticles may be woven together with strands of longer half-lives.

The osteoimplant may be machined or shaped by any suitable mechanicalshaping means. Computerized modeling may provide for theintricately-shaped three-dimensional architecture of an osteoimplantcustom-fitted to the bone repair site with precision.

Thus, the at least partially demineralized bone may optionally besubjected to a configuring step to form an implant. In an embodimentwherein the bone is compressed to enhance osteoinductivity, theconfiguring step may be done during compression. The configuring stepmay be employed using conventional equipment known to those skilled inthe art to produce a wide variety of geometries, e.g., concave or convexsurfaces, stepped surfaces, cylindrical dowels, wedges, blocks, screws,and the like. A surgically implantable material fabricated fromelongated bone particles that have been demineralized, which may beshaped as a sheet, and processes for fabricating shaped materials fromdemineralized bone particles are disclosed in U.S. Pat. Nos. 5,507,813and 6,436,138, respectively, the contents of which are incorporated byreference herein in their entireties. Suitable sheets include those soldunder the trade name Grafton® DBM Flex, which must be wetted/hydratedprior to use to be useful for implantation. Such sheets have recentlybeen reported as effective in seeding human bone marrow stromal cells(BMSCs), which may be useful in the repair of large bone defects. Kastenet al., Comparison of Human Bone Marrow Stromal Cells Seeded onCalcium-Deficient Hydroxyapatite, Betatricalcium Phosphate andDemineralized Bone Matrix, 24(15) Biomaterials 2593-2603 (2003). Alsouseful are demineralized bone and other matrix preparations comprisingadditives or carriers such as binders, fillers, plasticizers, wettingagents, surface active agents, biostatic agents, biocidal agents, andthe like. Some exemplary additives and carriers include polyhydroxycompounds, polysaccharides, glycosaminoglycan proteins, nucleic acids,polymers, polaxomers, resins, clays, calcium salts, and/or derivativesthereof. See, for example, U.S. Pat. No. 5,290,558, incorporated byreference herein in its entirety.

The bone used in creating the at least partially demineralized bone maybe obtained from any source of living or dead tissue. In many instances,the source of bone may be matched to the eventual recipient of theinventive composition. At a minimum, it is often desirable that thedonor and recipient are of the same species, though even xenogenicsources are permitted. Thus, for use in humans, generally DBM derived atleast in part from human bone may be used. For example, bone materialmay be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or morehuman bone material. In certain embodiments 100% of the bone material ishuman bone material.

In one embodiment, the osteoimplant induces endochondral bone formationreliably and reproducibly in a mammalian body. The at least partiallydemineralized bone osteoimplant comprises particles of porous materials.The pores must be of a dimension to permit progenitor cell migrationinto the carrier and subsequent differentiation and proliferation. Theparticle size may be within the range of 70 μm-850 μm, or 70 μm-420 μm,or 150 μm-420 μm, though any suitable size may be used. It may befabricated by close packing particulate material into a shape spanningthe bone defect, or by otherwise structuring as desired a material thatis biocompatible, and in some embodiments biodegradable in vivo to serveas a “temporary scaffold” and substratum for recruitment of migratoryprogenitor cells, and as a base for their subsequent anchoring andproliferation.

VI. Formulation

The osteoinductive at least partially demineralized bone or theosteoimplant may be formulated for a particular use. The formulation maybe used to alter the physical, biological, or chemical properties of theDBM. A physician would readily be able to determine the formulationneeded for a particular application, taking into account such factors asthe type of injury, the site of injury, the patient's health, and therisk of infection. In various embodiments, the at least partiallydemineralized bone may comprise, for example, less than approximately0.5% water, less than approximately 1% water, or less than approximately5% water.

The osteoinductive at least partially demineralized bone may beconfigured to be suturable. Thus, in certain embodiments, theosteoinductive at least partially demineralized bone or an osteoimplantformed from the osteoinductive at least partially demineralized bone maybe sutured in place in vivo.

An osteoimplant formed from osteoinductive cancellous DBM may beconfigured for expansion. Cancellous DBM has a sponge-like texture andexpands and conforms. Thus, an osteoimplant may be formed that is ableto expand and conform to a site in vivo. The osteoimplant so formed maycomprise monolithic cancellous bone or particulated (and reaggregated)cancellous bone.

Osteoinductive at least partially demineralized bone or osteoimplantsmay be prepared to have selected resorption/loss of osteoinductivityrates, or even to have different rates in different portions of animplant. For example, the formulation process may include the selectionof bone particles of a particular size or composition, combined with theselection of a particular stabilizing agent or agents and the amounts ofsuch agents.

Physical properties such as deformability and viscosity may also bechosen depending on the particular clinical application. The particlesof bone may be mixed with other materials and factors to improve othercharacteristics of the implant. For example, the at least partiallydemineralized bone material may be mixed with other agents to improvewound healing. These agents may include drugs, proteins, peptides,polynucleotides, solvents, chemical compounds, and biological molecules.

In another embodiment, an osteoimplant having a pre-selectedthree-dimensional shape is prepared by repeated application ofindividual layers of DBM, for example by 3-D printing as described byU.S. Pat. Nos. 5,490,962, 5,518,680, and 5,807,437, each incorporatedherein by reference in their entireties. Different layers may compriseindividual stabilized DBM preparations, or alternatively may compriseDBM layers treated with stabilizing agents after deposition of multiplelayers.

In the process of preparing improved inventive bone matrix materials,the materials may be produced entirely aseptically or be sterilized toeliminate any infectious agents such as HIV, hepatitis B, or hepatitisC. The sterilization may be accomplished using antibiotics, irradiation,chemical sterilization (e.g., ethylene oxide), super critical CO₂treatment, thermal sterilization, or combinations thereof. Other methodsknown in the art of preparing bone and cartilage matrices, such asdefatting, sonication, lyophilization, and combinations thereof may alsobe used. Since the biological activity of various materials includingdemineralized bone is known to be detrimentally affected by mostterminal sterilization processes, care must be taken when sterilizingthe inventive compositions. In some embodiments, the osteoimplantsdescribed herein will be prepared aseptically or sterilized.

VII. Optional Treatments

In other embodiments, the present invention provides methods ofincreasing the osteoinductivity of the at least partially demineralizedbone by further exposing the at least partially demineralized bone to atleast one treatment (e.g., a biological or chemical agent). In additionto treatment with collagenase (or other suitable compound) orcompaction, the at least partially demineralized bone may be exposed toa chemical or condition that selectively degrades inhibitors ofosteogenic activity and/or to a chemical or condition that activatesosteoinductive factors in the carrier. Thus, the resulting at leastpartially demineralized bone has an increased osteoinductivity,osteogenic, or chondrogenic activity compared to a similar at leastpartially demineralized bone not exposed to the treatment or condition,because inhibition of an osteoinductive, osteogenic, or chondrogenicfactor is blocked. In general, agents that inhibit or reduceosteoinductive, osteogenic, or chondrogenic activity may be referred toas bone/cartilage inhibitory factors (BCIF).

Generally, it may be desirable to remove the inhibitors quickly withoutdenaturing the osteoinductive factors. As will be appreciated by thoseskilled in the art, factors having osteoinductive, osteogenic, and/orchondrogenic activity may be inhibited by a variety of mechanismsincluding proteolytic degradation, binding or sequestration of thefactor, etc.

VIII. Optional Additives

Optionally, other additives may be included in the osteoinductive atleast partially demineralized bone. It will be appreciated that theamount of additive used will vary depending upon the type of additive,the specific activity of the particular additive preparation employed,and the intended use of the at least partially demineralized bone. Thedesired amount is readily determinable by the user. Any of a variety ofmedically and/or surgically useful optional substances may beincorporated in, or associated with, the osteoinductive factors eitherbefore, during, or after preparation of the at least partiallydemineralized bone.

In certain embodiments, the additive is adsorbed to or otherwiseassociated with the osteoimplant. The additive may be associated withthe osteoimplant through specific or non-specific interactions, orcovalent or noncovalent interactions. Examples of specific interactionsinclude those between a ligand and a receptor, an epitope and anantibody, etc. Examples of nonspecific interactions include hydrophobicinteractions, electrostatic interactions, magnetic interactions, dipoleinteractions, van der Waals interactions, hydrogen bonding, etc. Incertain embodiments, the additive is attached to the osteoimplant, forexample, to the carrier, using a linker so that the additive is free toassociate with its receptor or site of action in vivo. In otherembodiments the additive is either covalently or non-covalently attachedto the carrier. In certain embodiments, the additive may be attached toa chemical compound such as a peptide that is recognized by the DBM. Inanother embodiment, the additive is attached to an antibody, or fragmentthereof, that recognizes an epitope found within the DBM. An additivemay be provided within the osteoimplant in a sustained release format.For example, the additive may be encapsulated within biodegradablenanospheres, microspheres, or the like.

It will be understood by those skilled in the art that the lists ofoptional substances herewith included are not intended to be exhaustiveand that other materials may be admixed with bone-derived elementswithin the practice of the present invention.

Osteoinductive Factors

Osteoinductive factors may be added to the treated at least partiallydemineralized bone, thus further increasing the osteoinductivity of theat least partially demineralized bone. U.S. patent application Ser. No.11/555,608, entitled Bone Matrix Compositions and Methods, filed Nov. 1,2006, which is herein incorporated by reference in its entirety, teachessuitable methods for adding osteoinductive factors to the at leastpartially demineralized bone and for otherwise enhancing theosteoinductivity of the bone.

Osteoinductive factors include any agent that leads to or enhances theformation of bone. The osteoinductive factors may do this in any manner.For example, the osteoinductive factors may lead to the recruitment ofcells responsible for bone formation, the osteoinductive factors maylead to the secretion of matrix which may subsequently undergomineralization, the osteoinductive factors may lead to the decreasedresorption of bone, etc. Suitable osteoinductive factors include bonemorphogenic proteins (BMPs), transforming growth factor (TGF-0),insulin-like growth factor (IGF-1), parathyroid hormone (PTH), andangiogenic factors such as VEGF. In one embodiment, the osteoinductivefactors is genetically engineered to comprise an amino acid sequencewhich promotes the binding of the inducing agent to the DBM or thecarrier. Sebald et al., PCT/EP00/00637, incorporated herein by referencein its entirety, describe the production of exemplary engineered growthfactors suitable for use with DBM.

Other osteoinductive factors also may be added to the at least partiallydemineralized bone. These osteoinductive factors may be added in anactivated or nonactivated form, and they may be added at anytime duringthe preparation of the inventive material. For example, theosteoinductive factors may be added after the demineralization step andprior to the addition of the stabilizing agents so that the addedosteoinductive factors are protected from exogenous degrading enzymesonce implanted. In some embodiments, the at least partiallydemineralized bone is lyophilized in a solution containing theosteoinductive factors. In certain other embodiments, the osteoinductivefactors are adhered onto the hydrated demineralized bone matrix and arenot freely soluble. In other instances, the osteoinductive factors areadded after addition of a stabilizing agent so that the osteoinductivefactors are available immediately upon implantation of the at leastpartially demineralized bone.

Radiopaque Substances

Radiopaque substances may be added to impart radiopacity to the at leastpartially demineralized bone. Examples of substances impartingradiopacity include for example, fully mineralized bone particles,Barium- and Iodine-containing compounds or compositions, e.g., bariumsulfate and barium sulfate for suspension, lopanoic acid, and the like.When employed, substances imparting radiopacity will typically representfrom about 1 to about 25 weight percent of the bone particle containingcomposition, calculated prior to forming the shaped material. In someembodiments, the at least partially demineralized bone may have someinherent radiopacity due to remaining mineralization.

Angiogenesis Promoting Materials

Development of a vasculature around the implant site also may help formnew bone and/or cartilage tissues. Angiogenesis may be an importantcontributing factor for the replacement of new bone and cartilagetissues. In certain embodiments, angiogenesis is promoted so that bloodvessels are formed at the site to allow efficient transport of oxygenand other nutrients and growth factors to the developing bone orcartilage tissue. Thus, angiogenesis-promoting factors may be includedin the osteoimplant to increase angiogenesis in that region. Forexample, class 3 semaphorins, e.g., SEMA3, controls vascularmorphogenesis by inhibiting integrin function in the vascular system,Serini et al., 424 Nature 391-397 (2003), incorporated herein byreference in its entirety, and may be included in the osteoimplant.

Bioactive Agents

The osteoconductive at least partially demineralized bone may provide asystem for delivering bioactive agents, such as osteoinductive factors,to a host animal. Thus, the osteoimplant enables an improved healingresponse to the implant without the need to administer separately thebioactive agent. A problem with the introduction of the bioactive agentat the site is that it is often diluted and redistributed during thehealing process by the circulatory systems (e.g., blood, lymph) of therecipient before complete healing has occurred. A solution to thisproblem of redistribution is to affix the bioactive components to theosteoimplant. Some bioactive agents that may be delivered using an atleast partially demineralized bone composition include agents thatpromote the natural healing process, i.e., resorption, vascularization,angiogenesis, new growth, or the like. In one embodiment, theosteoimplant is provided in which the treated at least partiallydemineralized bone, together with a stabilizing agent, is used todeliver the biologically active agent. It is expected that thestabilizing agent will protect the biologically active agent fromdegradation, and therefore will extend its active life after deliveryinto the recipient animal. In certain embodiments, the bioactive agentis an osteoinductive agent, and in certain embodiments, the at leastpartially demineralized bone may be used to deliver more than onebioactive agent, more than two, and sometimes more than three bioactiveagents. The bioactive agent may be associated with the at leastpartially demineralized bone. For example, the bioactive agent may beassociated with the at least partially demineralized bone throughelectrostatic interactions, hydrogen bonding, pi stacking, hydrophobicinteractions, van der Waals interactions, etc. In certain embodiments,the bioactive agent is attached to the at least partially demineralizedbone through specific interactions such as those between a receptor andits ligand or between an antibody and its antigen. In other embodiments,the bioactive agent is attached to the at least partially demineralizedbone through non-specific interactions (e.g., hydrophobic interactions).

Medically/surgically useful substances include physiologically orpharmacologically active substances that act locally or systemically inthe host. Generally, these substances may include bioactive substanceswhich may be readily incorporated into the osteoimplant and include,e.g., demineralized bone powder as described in U.S. Pat. No. 5,073,373,the contents of which are incorporated herein by reference in itsentirety; collagen, insoluble collagen derivatives, etc., and solublesolids and/or liquids dissolved therein; antiviricides, particularlythose effective against HIV and hepatitis; antimicrobials and/orantibiotics such as erythromycin, bacitracin, neomycin, penicillin,polymycin B, tetracyclines, biomycin, chloromycetin, and streptomycins,cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentamycin,etc.; biocidal/biostatic sugars such as dextran, glucose, etc.; aminoacids; peptides; vitamins; inorganic elements; co-factors for proteinsynthesis; hormones; endocrine tissue or tissue fragments; synthesizers;enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases,etc.; polymer cell scaffolds with parenchymal cells; angiogenic agentsand polymeric carriers containing such agents; collagen lattices;antigenic agents; cytoskeletal agents; cartilage fragments; living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells; naturalextracts; genetically engineered living cells or otherwise modifiedliving cells; expanded or cultured cells; DNA delivered by plasmid,viral vectors or other means; tissue transplants; demineralized bonepowder; autogenous tissues such as blood, serum, soft tissue, bonemarrow, etc.; bioadhesives; BMPs; osteoinductive factor (IFO);fibronectin (FN); endothelial cell growth factor (ECGF); vascularendothelial growth factor (VEGF); cementum attachment extracts (CAE);ketanserin; human growth hormone (HGH); animal growth hormones;epidermal growth factor (EGF); interleukins, e.g., interleukin-1 (IL-1),interleukin-2 (IL-2); human alpha thrombin; transforming growth factor(TGF-beta); IGF-1 and IGF-2; platelet derived growth factors (PDGF);fibroblast growth factors (FGF, BFGF, etc.); periodontal ligamentchemotactic factor (PDLGF); enamel matrix proteins; growth anddifferentiation factors (GDF); hedgehog family of proteins; proteinreceptor molecules; small peptides derived from growth factors above;bone promoters; cytokines; somatotropin; bone digesters; antitumoragents; cellular attractants and attachment agents; immuno-suppressants;permeation enhancers, e.g., fatty acid esters such as laureate,myristate and stearate monoesters of polyethylene glycol, enaminederivatives, alpha-keto aldehydes, etc.; and nucleic acids. The amountsof such optionally added substances may vary widely with optimum levelsbeing readily determined in a specific case by routine experimentation.

In certain embodiments, the agent to be delivered is adsorbed to orotherwise associated with the osteoimplant. The agent may be associatedwith the osteoimplant through specific or non-specific interactions; orcovalent or non-covalent interactions. Examples of specific interactionsinclude those between a ligand and a receptor, an epitope and anantibody, etc. Examples of non-specific interactions include hydrophobicinteractions, electrostatic interactions, magnetic interactions, dipoleinteractions, van der Waals interactions, hydrogen bonding, etc. Incertain embodiments, the agent is attached to the osteoimplant using alinker so that the agent is free to associate with its receptor or siteof action in vivo. In certain embodiments, the agent to be delivered maybe attached to a chemical compound such as a peptide that is recognizedby the matrix of the at least partially demineralized bone composition.In another embodiment, the agent to be delivered is attached to anantibody, or fragment thereof, that recognizes an epitope found withinthe matrix of the DBM composition. In a further embodiment, the agent isa BMP, TGF-β, IGF, parathyroid hormone (PTH), growth factors, orangiogenic factors. In certain embodiments at least two bioactive agentsare attached to the at least partially demineralized bone composition.In other embodiments at least three bioactive agents are attached to theat least partially demineralized bone composition.

IX. Examples

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1

Preparation of Cancellous Dowels. Corticocancellous Dowels were drilledfrom the femoral condyle of a human donor using 12 mm Codman dowelcutter. Any attached cartilage or cortical bone was removed, leavingonly dense and diffuse cancellous structures. The bone was drilled whilein a frozen state and the drilled dowels were immediately placed in acold solution of 0.2 mM, 1 mM NaN₃, and 0.1 mM Benzamidine HCl (4° C.).The bones were removed after two hours on ice. In order to remove thebone marrow elements, the dowels were lavaged using semi-gentle(nondisruptive to cancellous structure) cool water. The material wasstored at −70° C. until defatting and demineralization.

Defatting and Demineralization. The dowels were defatted in a solutionof 1:1 chloroform:methanol for 15 hours at room temperature. Thesolution was then poured off and any organic residue was allowed toevaporate from the bone for 6 hours under a fume hood. The cancellousmatrix was demineralized in 0.6 N HCl at 4° C. overnight and washedextensively with water prior to lyophilization. Specifically, thefollowing protocol was followed:

-   -   1. Extraction with 400 ml 1:1 chloroform:methanol for 15 hours        at room temperature.    -   2. Evaporate 6 hours under fume hood.    -   3. 300 ml 0.6 N HCl at 4° C. overnight.    -   4. Change acid, remove air bubbles by pressing cancellous        structure, continue demineralization at 4° C. for 6 hours.    -   5. Wash with water using 800 ml at 4° C. Perform 4 times for 10        minutes each.    -   6. Lyophilize two dowels not treated with collagenase (one        large, one small) overnight—30, 23° C. 3 days. The untreated        dowels are control dowels.

Collagen Denaturation. The collagen structure of two dowels fortreatment with collagenase was gelatinized by the following sequence:

-   -   1. Extract 1 hour, 2 M CaCl₂ 4° C. (400 ml).    -   2. Wash 3×10 minutes using cold water.    -   3. Extract 1 hour 0.5 M EDTA pH 7.4 at 4° C. (400 ml).    -   4. Wash 3×10 minutes using cold water.    -   5. Extract 8 M LiCl overnight at 4° C. (400 ml).    -   6. Wash 1 hour using 400 ml water at 4° C.    -   7. Wash with 300 ml sterile water at 55° C. for 90 minutes.    -   8. Lyophilize after freezing.

The dowels so treated are referred to as bone matrix gelatin (BMG)samples.

Preparation of Samples. Each of the dowels were cut to produce 2 longDBM of approximately 100 mg each, 2 short DBM of approximately 50 mgeach, 2 long BMG of approximately 100 mg each, and 2 short BMG ofapproximately 60 mg each

Each sample was washed and rehydrated in enough Alloprep to cover thesample (approximately 750 μl).

Collagenase Treatment. Four samples were treated with collagenase. Twosamples were BMG samples. Two samples were DBM samples.

Materials:

-   -   Digestion buffer (50 mM Tris-HCl pH 7.4, containing 5 mM CaCl₂,        sterile filtered);    -   Chromatographically purified collagenase (Worthington CLSPA;        Source: Clostridium Histolyticum; 10,000 units); and    -   0.1N Glacial acetic acid (Add 248.56 ml of deionized water to        1.44 ml of 17.4 M Glacial Acetic Acid, Sterile filtered).

Generally, collagenase treatment comprised placing sections ofdemineralized cancellous dowels weighing between 50 and 100 mg in a 5 mldigestion buffer containing 80 units/ml collagenase. The samples wereincubated at 37° C. for 90 minutes. Each sample was washed for 30minutes with 30 ml 0.1N acetic acid in the cold. The samples were thenrinsed for 10 minutes with phosphate buffered saline (“PBS”) and 10minutes with deionized water prior to lyophilization. The specificprotocol was as follows:

-   -   1. Cut 2 cancellous BMG pieces, one from the BMG-long which        weighed 100 mg and one from the BMG-short which weighed 60 mg.    -   2. Cut 2 cancellous DBM pieces, one from the DBM-long which        weighed 100 mg and one from the DBM-short which weighed 50 mg.    -   3. Add 4.8 ml digestion buffer to each sample.    -   4. Add 200 μl of stock collagenase to each sample.    -   5. Vortex each sample.    -   6. Incubate each sample for 1.5 hours in a 37° C. water bath.    -   7. Add 30 ml 0.1N glacial acetic acid to each sample. Wash for 1        hour at 4° C. using a magnetic stir bar and plate.    -   8. Pour off the glacial acetic acid.    -   9. Wash with 45 ml PBS for 10 minutes at 4° C. Pour off PBS.        Repeat.    -   10. Wash with 45 ml deionized water for 10 minutes at 4° C. Pour        off deionized water.    -   11. Press samples into a syringe and freeze-dry using a 24 hour        cycle.

Implantation. The following pieces were implanted, aiming for a range of10-20 mg (all samples were implanted in athymic rats except for thosewhere mouse is indicated):

Collagenase short-BMG Collagenase long-BMG 9 mg 14 mg 8 mg 17 mg 12 mg18 mg 14 mg (mouse)

Collagenase short-DBM Collagenase long-DBM 7 mg 11 mg 9 mg 17 mg 18 mg18 mg 9 mg (mouse)

Control Short BMG Control Long BMG 10 mg 10 mg 11 mg 10 mg 12 mg 11 mg

Control Short DBM Control Long DBM 10 mg 10 mg 10 mg 11 mg 11 mg 12 mg

Assay. The osteoinductive potential of collagenase treated and untreated(control) cancellous bone samples was evaluated by implanting thematerials intramuscularly in athymic rats and an athymic mouse. After 28days the explants were evaluated histologically and radiographically forevidence of heterotopic bone formation.

Results. FIG. 1 is a graph illustrating the results of Example 1. Asshown, the collagenase treated DBM had higher histologic scores for boththe long and short samples than did the control DBM. The longcollagenase treated BMG sample had a lower histologic score than did thelong control BMG sample. The short collagenase treated BMG sample had ahigher histologic score than did the short control BMG sample.

The results demonstrate that collagenase treatment enhances theosteoinductive activity of cancellous DBM scaffolds. More specifically,the results indicate that the osteinductive activity of cancellous DBMscaffolds, as measured by the histologic score, was measurable, andincreased above that of untreated DBM, in all samples. The increase maybe noted both histologically and radiographically. Collagenase treatedhuman demineralized cancellous scaffolds have potent osteoinductiveactivity as measured by heterotopic bone formation in athymic rats. Thenegligible osteoinductive activity of human cancellous demineralizedscaffolds in athymic mice may be markedly enhanced by treatment withbacterial collagenase, without requiring treatment with LiCl prior totreatment with bacterial collagenase. The activity of these preparationsis comparable to that of human cortical DBM.

Example 2

Cancellous bone was demineralized in 0.6 N HCl at room temperature. Thedemineralized bone was washed, and the wet demineralized bone wassmeared to a consistency of a fibrous paste. The fibrous paste was thentreated using one of three methods:

-   -   1. Hand manipulated with slight pressure, dried at ambient        temperature for approximately 72 hours;    -   2. Placed in a PTFE mold and compressed between plungers. The        loaded hand clamped mold was placed in a vacuum oven and dried        at 40° C. for approximately 72 hours;    -   3. Loaded into a mold and compressed between stainless steel        dehydrothermal plungers. Initially, 500 pounds of force was        applied to the plungers to press out excess water and compress        the paste. After initial compression, dead weights were applied        to continue to keep the paste under compression while a vacuum        was drawn through the dehydrothermal plunters. During vacuum        drying (via the plungers), the mold was kept at approximately        40° C. for 72 hours.        After processing, each sample was placed in a beaker containing        room temperature deionized water, and observed for signs of        disassociation. FIG. 3 illustrates the formulations thus        produced. Formulation 200 corresponds to method 1, formulation        202 corresponds to method 2, and formulation 204 corresponds to        method 3. Each of the formulations 200, 202, and 204 were        submerged in deionized water and observed for cohesiveness. FIG.        4 illustrates formulation 200 at 7 hours of submersion in water.        FIG. 5 illustrates formulation 202 submerged in water for 72        hours. FIG. 6 illustrates formulation 204 submerged in water for        72 hours. From FIG. 4, formulation 200 swelled when submerged in        water but did not dissociate. From FIGS. 5 and 6, formulations        202 and 204 each generally retained their shape in water.

X. Assessment of Osteogenic Activity

Induction of bone formation may be determined by a histologicalevaluation showing the de novo formation of bone with accompanyingosteoblasts, osteoclasts, and osteoid matrix. For example,osteoinductive activity of an osteoinductive factor may be demonstratedby a test using a substrate onto which material to be tested isdeposited. The substrate with deposited material is implantedsubcutaneously in a test animal. The implant is subsequently removed andexamined microscopically for the presence of bone formation includingthe presence of osteoblasts, osteoclasts, and osteoid matrix. A suitableprocedure for assessing osteoinductive activity is illustrated inExample 5 of U.S. Pat. No. 5,290,763 (“the '763 patent”), hereinincorporated by reference in its entirety. Although there is nogenerally accepted scale of evaluating the degree of osteogenicactivity, certain factors are widely recognized as indicating boneformation. Such factors are referenced in the scale of 0-8 which isprovided in Table 3 of example 1 of U.S. Pat. No. 5,563,124, hereinincorporated by reference in its entirety. The 0-4 portion of this scalecorresponds to the scoring system described in the '763 patent, which islimited to scores of 0-4. The remaining portion of the scale, scores5-8, references additional levels of maturation of bone formation. Theexpanded scale also includes consideration of resorption of collagen, afactor which is not described in the '763 patent.

Human DBM has not been proven to have osteoinductive activity in higherorder species. Studies indicate that dog DBM is osteoinductive whentested in athymic rats but not in dogs. Further, studies have beenpublished stating that BMP-2 has osteoinductive activity in squirrelmonkeys but bone matrix does not. Collagenase treated DBM slowlysolubilizes in tissue culture media (in the presence of cells) at 37° C.Without being bound by theory, it is possible that the lack ofosteoinductive activity of DBM in higher species may be related to thefailure of DBM to present growth factors to the host in an efficientmanner. Collagenase treatment provides a DBM capable of osteoinductiveactivity in higher species. Such formation may serve as a basis foreliminating the need for autograft in many orthopaedic applications,including posterior spinal fusion.

XI. Uses

The osteogenic osteoimplant is intended to be applied at a bone repairsite, for example, a site resulting from injury, defect brought aboutduring the course of surgery, infection, malignancy, or developmentalmalformation. The osteoimplant may be utilized in a wide variety oforthopaedic, periodontal, neurosurgical, and oral and maxillofacialsurgical procedures.

Demineralized cancellous bone has a sponge-like structure. The materialmay be compressed significantly and will substantially regain itsoriginal structure in the absence of the compressive force. Anosteoinductive cancellous scaffold may be forced into a space or defectthrough a small opening. The structure will then expand and conform tothe shape of the environment. This characteristic may be particularlyuseful in embodiments wherein the collagenous structure has beenaltered, for example, by treatment with collagenase. Thus, in oneapplication, an osteoinductive cancellous scaffold is placed into acompromised vertebral element in a procedure similar to vertebroplasty.It is expected that the cancellous scaffold would expand in thecollapsed vertebrae and, over time, new bone formed in the interior ofthe vertebral element would restore its structural integrity. Theprocedure could be used in conjunction with or as a replacement forKyphoplasty.

In addition or alternatively, demineralized cancellous bone may beground into a particulate and formed into a compressed implant having adenser structure compared to the implant described above. The denseimplant may be used alone or in combination with implant scaffolding.The implant may expand in vivo over the course of between a few hours toa few weeks depending on, for example, the density of the implant, othermaterials incorporated into the compressed implant, the size and shapeof the implant, or other aspects of the implant that would be readilyunderstandable by those skilled in the art.

At the time just prior to when the osteoimplant of the invention is tobe placed in a defect site, optional materials, e.g., autograft bonemarrow aspirate, autograft bone, preparations of selected autograftcells, autograft cells containing genes encoding bone promoting action,etc., may be combined with the osteoimplant. The osteoimplant may beimplanted at the bone repair site, if desired, using any suitableaffixation means, e.g., sutures, staples, bioadhesives, screws, pins,rivets, other fasteners and the like, or it may be retained in place bythe closing of the soft tissues around it.

Activated scaffolds comprising the osteoinductive cancellous DBM mayreduce or eliminate the need for harvesting iliac crest autograft inchallenging orthopaedic applications such as posterolateral spinalfusion and the treatment of critical sized nonunion fractures.

The osteoinductive at least partially demineralized bone may also beused as a drug delivery device. Demineralized cancellous bone has thecapacity to absorb significant amounts of fluids, and it has a highsurface area to volume ratio. These properties indicate that thematerial would be an ideal growth scaffold for bioactive cells.Osteoinductive at least partially demineralized bone may be used as acarrier for osteogenic cells, chrondrogenic cells, stem cells, bonemarrow aspirates, fat cells, and other cell types that are involved intissue formation or that have the capability to differentiate intotissue forming cells when exposed to appropriate signals. The at leastpartially demineralized bone may also serve as a carrier for a varietyof native or recombinant growth and differentiation factors.

Collagenase treatment of at least partially demineralized bone may havefurther applications. For example, cells harvested from a recipient maybe treated with collagenase treated at least partially demineralizedbone in a manner that induces their differentiation into bone,cartilage, or other types of tissues such as bone marrow, neural cells,or vasculature. The tissues/organs could then be transplanted into therecipient after they have developed to a certain point in vitro. Inaddition, at least partially demineralized bone contains a variety ofgrowth factors. The type of growth factors harvested, and themanipulation of cell culture conditions or specific growth factor levelsin at least partially demineralized bone, may be optimized for thetissue of interest.

In certain embodiments, association with the osteoinductive at leastpartially demineralized bone increases the half-life of the relevantbiologically active agent(s). Certain inventive drug delivery devicesare used to deliver osteoinductive growth factors. Other agents that maybe delivered include factors or agents that promote wound healing.However, the osteoinductive at least partially demineralized bone mayalternatively or additionally be used to deliver other pharmaceuticalagents including antibiotics, anti-neoplastic agents, growth factors,hematopoietic factors, nutrients, an other bioactive agents describedabove. The amount of the bioactive agent included with the at leastpartially demineralized bone composition can vary widely and will dependon such factors as the agent being delivered, the site ofadministration, and the patient's physiological condition. The optimumlevels is determined in a specific case based upon the intended use ofthe implant.

XII. Conclusion

In certain embodiments, the osteoinductive at least partiallydemineralized bone and associated osteoimplants produce bone orcartilage in an animal model and/or in human patients with similartiming and at a level at least 10%, 20%, 35%, 50%, 100%, 200%, 300%, or400% or greater osteogenic, osteoinductive or chondrogenic activity thana corollary at least partially demineralized bone that has not beenexposed to a treatment or condition as described herein. Of course, oneskilled in the art will appreciate that these values may vary dependingon the type of test used to measure the osteoinductivity or osteogenicor chondrogenic activity described above. The test results may fallwithin the range of 10% to 35%, 35% to 50%, 50% to 100%, 100% to 200%,and 200% to 400%. In certain embodiments, when an osteoimplant isimplanted into a bone defect site, the osteoimplant has anosteoinductivity score of at least 1, 2, 3, or 4 in an animal modeland/or in humans. In some embodiments, when an osteoimplant comprisingosteoinductive cancellous DBM is implanted in a bone defect site, theosteoimplant has an osteoinductivity score comparable to a cortical DBMimplant that has not been treated.

Although the invention has been described with reference to variousembodiments, persons skilled in the art will recognize that changes maybe made in form and detail without departing from the spirit and scopeof the invention.

1-72. (canceled)
 73. An osteoinductive composition comprising: at leastpartially demineralized cancellous bone, the at least partiallydemineralized cancellous bone having been treated to increase theosteoinductive activity of the bone; wherein the at least partiallydemineralized cancellous bone exhibits increased osteoinductive activitywhen compared to untreated at least partially demineralized cancellousbone.
 74. The osteoinductive composition of claim 73, wherein thecomposition has a histologic osteoinductivity score of at least 1 on a 4point scale.
 75. The osteoinductive composition of claim 73, wherein thecomposition induces bone formation in higher order species.
 76. Theosteoinductive composition of claim 73, wherein the composition exhibitsa solid structure at room temperature and substantially liquefies whenimplanted in a body.
 77. The osteoinductive composition of claim 73,wherein the composition has enhanced solubility when compared tosolubility of untreated at least partially demineralized bone matrix.78. The osteoinductive composition of claim 73, wherein trabecularstructure of the treated at least partially demineralized cancellousbone is compacted when compared to trabecular structure of untreated atleast partially demineralized cancellous bone.
 79. The osteoinductivecomposition of claim 73, wherein the at least partially demineralizedcancellous bone exhibits a trabecular density that is increased whencompared to untreated at least partially demineralized cancellous bone.80. The osteoinductive composition of claim 73, wherein collagen of thetreated at least partially demineralized cancellous bone is disruptedwhen compared to collagen of untreated at least partially demineralizedcancellous bone.
 81. The osteoinductive composition of claim 73, whereina portion of collagen present in the treated at least partiallydemineralized cancellous bone is present as collagen fragments.
 82. Theosteoinductive composition of claim 73, wherein the at least partiallydemineralized cancellous bone is compacted and wherein the compositionretains its shape in water for a predetermined period of time.
 83. Theosteoinductive composition of claim 73, wherein the at least partiallydemineralized cancellous bone is particulated and reformed into animplant.
 84. The osteoinductive composition of claim 83, wherein theimplant further comprises a carrier.
 85. The osteoinductive compositionof claim 84, wherein the carrier is a polymer and wherein the cancellousbone is treated after blending with the polymer.
 86. The osteoinductivecomposition of claim 73, wherein the at least partially demineralizedcancellous bone is compacted.
 87. The osteoinductive composition ofclaim 73, further comprising at least partially demineralized corticalbone that has been treated to increase the osteoinductive activity ofthe bone.
 88. The osteoinductive composition of claim 73, furthercomprising at least partially demineralized corticocancellous bone thathas been treated to increase the osteoinductive activity of the bone.89. The osteoinductive composition of claim 73, further comprising abioactive agent.
 90. The osteoinductive composition of claim 73, whereinnative inductive materials of the at least partially demineralized bonematrix are substantially exposed.
 91. The osteoinductive composition ofclaim 73, further comprising at least one inductive material.
 92. Theosteoinductive composition of claim 73, wherein one or more structuralcomponents of the treated at least partially demineralized bone matrixis altered. 93-184. (canceled)